Poldip2 controls leukocyte infiltration into the ischemic brain by regulating focal adhesion kinase-mediated VCAM-1 induction.

Stroke is a multiphasic process involving a direct ischemic brain injury which is then exacerbated by the influx of immune cells into the brain tissue. Activation of brain endothelial cells leads to the expression of adhesion molecules such vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells, further increasing leukocyte recruitment. Polymerase δ-interacting protein 2 (Poldip2) promotes brain vascular inflammation and leukocyte recruitment via unknown mechanisms. This study aimed to define the role of Poldip2 in mediating vascular inflammation and leukocyte recruitment following cerebral ischemia. Cerebral ischemia was induced in Poldip2+/+ and Poldip2+/- mice and brains were isolated and processed for flow cytometry or RT-PCR. Cultured rat brain microvascular endothelial cells were used to investigate the effect of Poldip2 depletion on focal adhesion kinase (FAK)-mediated VCAM-1 induction. Poldip2 depletion in vivo attenuated the infiltration of myeloid cells, inflammatory monocytes/macrophages and decreased the induction of adhesion molecules. Focusing on VCAM-1, we demonstrated mechanistically that FAK activation was a critical intermediary in Poldip2-mediated VCAM-1 induction. In conclusion, Poldip2 is an important mediator of endothelial dysfunction and leukocyte recruitment. Thus, Poldip2 could be a therapeutic target to improve morbidity following ischemic stroke.

Poldip2 deficiency protects against lung edema and vascular inflammation in a model of acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) in a deadly disease that can be brought on by endotoxins such as lipopolysaccharide (LPS). ARDS is characterized by vascular permeability, a severe inflammatory response, lung leukocyte infiltration, and resultant lung edema. Polymerase δ-interacting protein 2 (Poldip2) is a novel regulator of blood-brain barrier permeability; however, its role in regulating lung permeability and vascular inflammation is unknown. Here, the role of Poldip2 in regulating vascular permeability and inflammation in a mouse model of ARDS was assessed. Heterozygous deletion of Poldip2 was found to reduce LPS-induced mortality within 20 h, lung inflammatory signaling, and leukocyte infiltration. Moreover, reduced Poldip2-suppressed LP-induced vascular cell adhesion molecule (VCAM)-1 induction, leukocyte recruitment, and mitochondrial reactive oxygen species (ROS) production in vitro These data indicate that Poldip2 is an important regulator of the debilitating consequences of ARDS, potentially through the regulation of mitochondrial ROS-induced inflammatory signaling. Consequently, inhibition of Poldip2 may be a viable option for therapeutic discovery moving forward.

NOX4 (NADPH Oxidase 4) and Poldip2 (Polymerase δ-Interacting Protein 2) Induce Filamentous Actin Oxidation and Promote Its Interaction With Vinculin During Integrin-Mediated Cell Adhesion.

Objective- Actin cytoskeleton assembly and organization, as a result of focal adhesion (FA) formation during cell adhesion, are dependent on reactive oxygen species and the cellular redox environment. Poldip2 (polymerase δ-interacting protein 2), a novel regulator of NOX4 (NADPH oxidase 4), plays a significant role in reactive oxygen species production and cytoskeletal remodeling. Thus, we hypothesized that endogenous reactive oxygen species derived from Poldip2/NOX4 contribute to redox regulation of actin and cytoskeleton assembly during integrin-mediated cell adhesion. Approach and Results- Using vascular smooth muscle cells, we verified that hydrogen peroxide (H2O2) levels increase during integrin-mediated cell attachment as a result of activation of NOX4. Filamentous actin (F-actin) was oxidized by sulfenylation during cell attachment, with a peak at 3 hours (0.80±0.04 versus 0.08±0.13 arbitrary units at time zero), which was enhanced by overexpression of Poldip2. Depletion of Poldip2 or NOX4 using siRNA, or scavenging of endogenous H2O2 with catalase, inhibited F-actin oxidation by 78±26%, 99±1%, and 98±1%, respectively. To determine the consequence of F-actin oxidation, we examined the binding of F-actin to vinculin, a protein involved in FA complexes that regulates FA maturation. Vinculin binding during cell adhesion as well as migration capacity were inhibited after transfection with actin containing 2 oxidation-resistant point mutations (C272A and C374A). Silencing of Poldip2 or NOX4 also impaired actin-vinculin interaction, which disturbed maturation of FAs and inhibited cell migration. Conclusions- These results suggest that integrin engagement during cell attachment activates Poldip2/Nox4 to oxidize actin, which modulates FA assembly.

Poldip2 is an oxygen-sensitive protein that controls PDH and αKGDH lipoylation and activation to support metabolic adaptation in hypoxia and cancer.

Although the addition of the prosthetic group lipoate is essential to the activity of critical mitochondrial catabolic enzymes, its regulation is unknown. Here, we show that lipoylation of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (αKDH) complexes is a dynamically regulated process that is inhibited under hypoxia and in cancer cells to restrain mitochondrial respiration. Mechanistically, we found that the polymerase-δ interacting protein 2 (Poldip2), a nuclear-encoded mitochondrial protein of unknown function, controls the lipoylation of the pyruvate and α-KDH dihydrolipoamide acetyltransferase subunits by a mechanism that involves regulation of the caseinolytic peptidase (Clp)-protease complex and degradation of the lipoate-activating enzyme Ac-CoA synthetase medium-chain family member 1 (ACSM1). ACSM1 is required for the utilization of lipoic acid derived from a salvage pathway, an unacknowledged lipoylation mechanism. In Poldip2-deficient cells, reduced lipoylation represses mitochondrial function and induces the stabilization of hypoxia-inducible factor 1α (HIF-1α) by loss of substrate inhibition of prolyl-4-hydroxylases (PHDs). HIF-1α-mediated retrograde signaling results in a metabolic reprogramming that resembles hypoxic and cancer cell adaptation. Indeed, we observe that Poldip2 expression is down-regulated by hypoxia in a variety of cell types and basally repressed in triple-negative cancer cells, leading to inhibition of lipoylation of the pyruvate and α-KDH complexes and mitochondrial dysfunction. Increasing mitochondrial lipoylation by forced expression of Poldip2 increases respiration and reduces the growth rate of cancer cells. Our work unveils a regulatory mechanism of catabolic enzymes required for metabolic plasticity and highlights the role of Poldip2 as key during hypoxia and cancer cell metabolic adaptation.

Angiogenin in age-related macular degeneration.

PURPOSE: Age-related macular degeneration (AMD) is a common blinding disease in the elderly population. AMD is frequently complicated by choroidal neovascularization, causing irreversible losses in visual acuity. Proteins that induce pathologic angiogenesis in other systems include angiogenin, a small protein involved in angiogenesis in tumor metastases. Our goal was to determine if angiogenin participates in angiogenesis during choroidal neovascular membrane formation in AMD. METHODS: The expression of angiogenin in the human retina and retinal pigment epithelium (RPE)-choroid was determined using reverse-transcription (RT)-PCR and immunoblotting. Localization of angiogenin in human control eyes and in eyes with choroidal neovascularization was determined using immunohistochemistry. Potential angiogenin-mediated effects on endothelial cell migration, as well as angiogenin internalization by Rf/6a cells, were determined. RESULTS: Angiogenin was synthesized by the human choroid and retina and localized to normal and pathologic vasculature. Angiogenin did not change the migratory behavior of Rf/6a chorioretinal endothelial cells; however, these cells did internalize exogenous angiogenin in culture. CONCLUSIONS: Chorioretinal endothelial cells bind and internalize angiogenin, a protein localized to the choroid in normal eyes, as well as in some drusen and in neovascular membranes in AMD eyes. Angiogenin has been shown to participate in angiogenesis in other tissues. Although angiogenin does not increase the migratory behavior of these cells, it may play a role in other aspects of endothelial cell activation in neovascular AMD.

Choriocapillaris vascular dropout related to density of drusen in human eyes with early age-related macular degeneration.

PURPOSE: Age-related macular degeneration (AMD) is a common, potentially blinding disease characterized by the presence of extracellular deposits beneath the retinal pigment epithelium (RPE). Choroidal vascular changes have also been noted in AMD. This study examined the relationship between the choroidal vasculature and extent of drusen and other sub-RPE deposits, the key pathologic landmarks of AMD. METHODS: Sections of the maculas of 45 human eyes (21 early AMD and 24 age-matched control) were evaluated morphometrically. The cross-sectional area of sub-RPE deposits, vascular density, number of CD45+ leukocytes, and number of "ghost vessels" were determined in a masked fashion and evaluated by regression analysis. In addition, the extramacular vascular density either directly beneath drusen or adjacent to drusen was evaluated in a separate set of donor eyes. RESULTS: The vascular density of the choriocapillaris showed a trend toward decreasing in association with AMD status. By linear regression analysis, vascular density was inversely associated with sub-RPE deposit density (r(2) = 0.22, P < 0.01). The number of ghost vessels was negatively correlated with vascular density (r(2) = 0.55, P < 0.001) and positively correlated with sub-RPE deposit density (r(2) = 0.57, P < 0.001). In morphologic studies of extramacular solitary drusen, vascular density beneath drusen was found to be 45% lower than adjacent to drusen (P < 0.01). CONCLUSIONS: These findings support the concept that microvascular changes are related to the pathogenesis of AMD and suggest that vascular endothelial cell loss occurs in association with sub-RPE deposit formation. Whether microvascular events are a cause or consequence of drusen or other deposit formation remains to be determined.

Anti-γ-enolase autoimmune retinopathy manifesting in early childhood.

OBJECTIVE: To describe the clinical, molecular, and serologic findings of a case in which autoimmune retinopathy and early-onset heritable retinal degeneration were both considered in the differential diagnosis. METHODS: A 3-year-old girl had clinical findings suggestive of a childhood-onset retinal degeneration. Samples of DNA and serum were collected. The coding regions of 11 genes associated with Leber congenital amaurosis were sequenced. The patient's serum reactivity to soluble and insoluble fractions of human retinal protein was compared with that of healthy control subjects (n = 32), patients with inflammatory eye disease (n = 80), and patients with molecularly confirmed retinal degenerations (n = 11). Two-dimensional gel electrophoresis and mass spectrometry were used to identify a protein that corresponded to a reactive band on Western blot. RESULTS: No plausible disease-causing mutations were identified in any of the retinal disease genes tested. However, the patient's serum showed reactivity to a single retinal antigen of approximately 47 kDa. Two-dimensional gel electrophoresis and mass spectrometry revealed the major reactive species to be neuron-specific enolase (NSE). Autoantibodies targeting NSE were not observed in any healthy control subjects or patients with inflammatory eye disease. However, anti-NSE activity was found in 1 child with molecularly confirmed Leber congenital amaurosis. CONCLUSION: This patient's clinical and laboratory findings coupled with the recently discovered role of anti-NSE antibodies in canine autoimmune retinopathy suggest that autoantibodies targeting NSE are involved in the pathogenesis of her disease. CLINICAL RELEVANCE: Infection or inflammation within the retina early in life may lead to an autoimmune phenocopy of early-onset inherited retinal degeneration.

Localization of complement 1 inhibitor (C1INH/SERPING1) in human eyes with age-related macular degeneration.

Age-related macular degeneration (AMD) is a common degenerative disease resulting in injury to the retina, retinal pigment epithelium and choriocapillaris. Recent data from histopathology, animal models and genetic studies have implicated altered regulation of the complement system as a major factor in the incidence and progression of this disease. A variant in the gene SERPING1, which encodes C1INH, an inhibitor of the classical and lectin pathways of complement activation, was recently shown to be associated with AMD. In this study we sought to determine the localization of C1INH in human donor eyes. Immunofluorescence studies using a monoclonal antibody directed against C1INH revealed localization to photoreceptor cells, inner nuclear layer neurons, choriocapillaris, and choroidal extracellular matrix. Drusen did not exhibit labeling. Genotype at rs2511989 did not appear to affect C1INH abundance or localization, nor was it associated with significant molecular weight differences when evaluated by Western blot. In a small number of eyes (n = 7 AMD and n = 7 control) AMD affection status was correlated with increased abundance of choroidal C1INH. These results indicate that C1INH protein is present in the retina and choroid, where it may regulate complement activation.

Differential macular and peripheral expression of bestrophin in human eyes and its implication for best disease.

PURPOSE: Best disease, or vitelliform macular degeneration, is an autosomal dominant form of macular degeneration that is caused by mutations in the gene encoding bestrophin. On clinical examination, Best disease is characterized by an elevated lesion beneath the neurosensory retina, resembling an egg yolk. The lesions in Best disease are primarily restricted to the macula, a small region of the retina responsible for central vision. The nature of the vitelliform material and the reason the development of such lesions is usually restricted to the macula are two unsolved questions in the pathogenesis of this disorder. METHODS: The expression of bestrophin protein and mRNA was evaluated by immunohistochemistry, Western blot, and quantitative PCR in a series of normal human eyes. The ultrastructure of the retinal pigment epithelium and the histopathology of two donors with clinically diagnosed Best disease were also examined. RESULTS: An eye from a Best disease donor with a T6R mutation was found to have deposits containing lipid and glycoconjugates within the central retinal scar. These deposits may be remnants of the vitelliform lesion. Immunohistochemical localization of bestrophin in a series of 22 unaffected eyes revealed a pattern in which macular labeling was less robust than labeling outside the macula in most (18/22) cases. This pattern was confirmed using quantitative PCR and Western blotting. CONCLUSIONS: Topographic differences in the levels of bestrophin protein may in part explain the propensity for the macula to develop lesions in Best disease.