Adaptive Cerebral Neovascularization in a Model of Type 2 Diabetes: Relevance to Focal Cerebral Ischemia

OBJECTIVE

The effect of diabetes on neovascularization varies between different organ systems. While excessive angiogenesis complicates diabetic retinopathy, impaired neovascularization contributes to coronary and peripheral complications of diabetes. However, how diabetes influences cerebral neovascularization is not clear. Our aim was to determine diabetes-mediated changes in the cerebrovasculature and its impact on the short-term outcome of cerebral ischemia.

RESEARCH DESIGN AND METHODS

Angiogenesis (capillary density) and arteriogenesis (number of collaterals and intratree anostomoses) were determined as indexes of neovascularization in the brain of control and type 2 diabetic Goto-Kakizaki (GK) rats. The infarct volume, edema, hemorrhagic transformation, and short-term neurological outcome were assessed after permanent middle–cerebral artery occlusion (MCAO).

RESULTS

The number of collaterals between middle and anterior cerebral arteries, the anastomoses within middle–cerebral artery trees, the vessel density, and the level of brain-derived neurotrophic factor were increased in diabetes. Cerebrovascular permeability, matrix metalloproteinase (MMP)-9 protein level, and total MMP activity were augmented while occludin was decreased in isolated cerebrovessels of the GK group. Following permanent MCAO, infarct size was smaller, edema was greater, and there was no macroscopic hemorrhagic transformation in GK rats.

CONCLUSIONS

The augmented neovascularization in the GK model includes both angiogenesis and arteriogenesis. While adaptive arteriogenesis of the pial vessels and angiogenesis at the capillary level may contribute to smaller infarction, changes in the tight junction proteins may lead to the greater edema following cerebral ischemia in diabetes.Type 2 diabetic patients hold two- to fourfold higher risk for cerebrovascular disease and stroke (1,2), and 70% of patients with a recent stroke have overt diabetes or pre-diabetes distinguished by impaired fasting glucose or impaired glucose tolerance (3). However, the underlying basis of this increased risk remains unclear. Diabetic vascular complications are well studied in diabetic retinopathy, nephropathy, peripheral arterial disease, and coronary artery disease. However, diabetes-induced structural and functional changes in the cerebral vasculature are unknown.It is becoming clear that the integrity of cerebral blood vessels is critical in the pathophysiology of stroke. While type 2 diabetes accounts for ∼90–95% of all diagnosed cases of diabetes in adult patients, most of the experimental studies are focused on type 1 diabetes induced by streptozotocin (STZ) injection, which is associated with high-level and short-term elevations of blood glucose. We have recently shown that diabetic Goto-Kakizaki (GK) rats develop smaller infarct and greater hemorrhagic transformation after stroke. These animals also showed cerebrovascular remodeling characterized by increased vessel tortuosity, vascular endothelial growth factor (VEGF) expression, and matrix metalloproteinase (MMP)-2 and -9 activity (4). Generated from glucose-intolerant Wistar rats, GK rat is a nonobese model of spontaneous type 2 diabetes with moderately elevated glucose levels (5,6). In light of previous reports that showed greater ischemic damage in acute hyperglycemic models of stroke (7–9), we hypothesized that chronic moderate hyperglycemia as seen in the GK model promotes neovascularization that affects the extent of ischemic injury. Considering that neovascularization may originate from new vessel formation as well as functional remodeling of existing vessels, the goals of the current study were 1) to determine the effect of diabetes on capillary density and arteriogenesis, 2) to determine the influence of diabetes on blood-brain barrier (BBB) baseline permeability and expression of proteins important for vascular integrity, and finally 3) to evaluate the effect of ischemic injury on infarct and hemorrhagic transformation development in diabetic rats with preexisting vascular disease, all of which may impact the outcome of ischemic injury.     

Rituximab Therapy in Idiopathic Membranous Nephropathy: A 2-Year Study

Background and objectives: It was postulated that in patients with membranous nephropathy (MN), four weekly doses of Rituximab (RTX) would result in more effective B cell depletion, a higher remission rate, and maintaining the same safety profile compared with patients treated with RTX dosed at 1 g every 2 weeks. This hypothesis was supported by previous pharmacokinetic (PK) analysis showing that RTX levels in the two-dose regimen were 50% lower compared with nonproteinuric patients, which could potentially result in undertreatment.Design, setting, participants, & measurements: Twenty patients with MN and proteinuria >5 g/24 h received RTX (375 mg/m² × 4), with re-treatment at 6 months regardless of proteinuria response. PK analysis was conducted simultaneously with immunological analyses of T and B cells to ascertain the effect of RTX on lymphocyte subpopulations.Results: Baseline proteinuria of 11.9 g/24 h decreased to 4.2 and 2.0 g/24 h at 12 and 24 months, respectively, whereas creatinine clearance increased from 72.4 ml/min per 1.73 m² at baseline to 88.4 ml/min per 1.73 m² at 24 months. Of 18 patients who completed 24-month follow-up, 4 are in complete remission, 12 are in partial remission, 1 has a limited response, and 1 patient relapsed. Serum RTX levels were similar to those obtained with two doses of RTX.Conclusions: Four doses of RTX resulted in more effective B cell depletion, but proteinuria reduction was similar to RTX at 1 g every 2 weeks. Baseline quantification of lymphocyte subpopulations did not predict response to RTX therapy.We have previously reported that in patients with idiopathic membranous nephropathy (MN), treatment with Rituximab (RTX; 1 g, intravenous on days 1 and 15) leads to complete (CR) or partial remission (PR) of proteinuria in 60% of patients at 12 months (1). However, pharmacokinetic (PK) analysis suggested that drug exposure may not have been optimal because RTX levels were 50% of those observed in rheumatoid arthritis (RA), a group with no proteinuria, resulting in a faster B cell recovery in patients with MN (1). On the basis of these findings, we conducted a study postulating that RTX given according to the lymphoma protocol (four weekly doses of 375 mg/m² each), with re-treatment at 6 months, would result in a more effective and prolonged B cell depletion and a higher remission rate while maintaining a similar safety profile. At the same time, a detailed PK analysis was repeated to assess the drug exposure question. We also measured human anti-chimeric antibodies (HACAs) because development of these antibodies may affect the B cell depletion after RTX and increase the risk for side effects (2).In patients with systemic lupus erythematosus (SLE), RA, and anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV), several abnormalities in peripheral B cells subsets have been described (3–5). In patients with SLE, B cell subset anomalies resolved after RTX therapy in parallel with clinical improvement (6). Similarly, in patients with RA, analysis of B cell subsets correlates with efficacy of response and the likelihood of relapse (4,7). In MN, experimental data suggest that B cells are involved in the pathogenesis of the disease (8). To date, the best proven therapy for patients with MN consists of the combined use of corticosteroids and cyclophosphamide (CYC). Because the mechanism of action of CYC includes suppression of various stages of the B cell cycle, including B cell activation, proliferation, and differentiation and inhibition of Ig secretion, it lends credence to the hypothesis that B cell abnormalities are involved in the pathogenesis of MN (9,10). However, there is a paucity of information on B cell subpopulations in patients with MN and on the characteristics and kinetics of B cell repopulation after RTX treatment in these patients.The data on T cell immunophenotyping in MN patients are sparse, but a high CD4⁺/CD8⁺ T cell ratio has been reported as predictive of response to treatment (11). In another study of MN patients, Kuroki et al. demonstrated that T and B cell dysregulation results in Th2 predominance and appropriate cytokine secretion with a concomitant increase in production of IgG4 by B cells (12). Thus, because the mechanism of action of RTX in MN is unknown and response is incomplete (only two-thirds of patients respond), we conducted a systematic and serial evaluation of B and T cells before RTX and at defined intervals after treatment to determine whether baseline values or the kinetics of subpopulation recovery in B and/or T cells influenced clinical outcome.     

Metformin Treatment in the Period After Stroke Prevents Nitrative Stress and Restores Angiogenic Signaling in the Brain in Diabetes

Diabetes impedes vascular repair and causes vasoregression in the brain after stroke, but mechanisms underlying this response are still unclear. We hypothesized that excess peroxynitrite formation in diabetic ischemia/reperfusion (I/R) injury inactivates the p85 subunit of phosphoinositide 3-kinase (PI3K) by nitration and diverts the PI3K–Akt survival signal to the p38–mitogen-activated protein kinase apoptosis pathway. Nitrotyrosine (NY), Akt and p38 activity, p85 nitration, and caspase-3 cleavage were measured in brains from control, diabetic (GK), or metformin-treated GK rats subjected to sham or stroke surgery and in brain microvascular endothelial cells (BMVECs) from Wistar and GK rats subjected to hypoxia/reoxygenation injury. GK rat brains showed increased NY, caspase-3 cleavage, and p38 activation and decreased Akt activation. Metformin attenuated stroke-induced nitrative signaling in GK rats. GK rat BMVECs showed increased basal nitrative stress compared with controls. A second hit by hypoxia/reoxygenation injury dramatically increased the nitration of p85 and activation of p38 but decreased Akt. These effects were associated with impairment of angiogenic response and were restored by treatment with the peroxynitrite scavenger 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride or the nitration inhibitor epicatechin. Our results provide evidence that I/R-induced peroxynitrite inhibits survival, induces apoptosis, and promotes peroxynitrite as a novel therapeutic target for the improvement of reparative angiogenesis after stroke in diabetes.     

Heme-regulated eIF2α kinase activated Atf4 signaling pathway in oxidative stress and erythropoiesis

Heme-regulated eIF2α kinase (Hri) is necessary for balanced synthesis of heme and globin. In addition, Hri deficiency exacerbates the phenotypic severity of β-thalassemia intermedia in mice. Activation of Hri during heme deficiency and in β-thalassemia increases eIF2α phosphorylation and inhibits globin translation. Under endoplasmic reticulum stress and nutrient starvation, eIF2α phosphorylation also induces the Atf4 signaling pathway to mitigate stress. Although the function of Hri in regulating globin translation is well established, its role in Atf4 signaling in erythroid precursors is not known. Here, we report the role of the Hri-activated Atf4 signaling pathway in reducing oxidative stress and in promoting erythroid differentiation during erythropoiesis. On acute oxidative stress, Hri(-/-) erythroblasts suffered from increased levels of reactive oxygen species (ROS) and apoptosis. During chronic iron deficiency in vivo, Hri is necessary both to reduce oxidative stress and to promote erythroid differentiation. Hri(-/-) mice developed ineffective erythropoiesis during iron deficiency with inhibition of differentiation at the basophilic erythroblast stage. This inhibition is recapitulated during ex vivo differentiation of Hri(-/-) fetal liver erythroid progenitors. Importantly, the Hri-eIF2αP-Atf4 pathway was activated and required for erythroid differentiation. We further demonstrate the potential of modulating Hri-eIF2αP-Atf4 signaling with chemical compounds as pharmaceutical therapies for β-thalassemia.     

Immune Reconstitution after Hematopoietic Stem Cell Transplantation in Immunodeficiency–Centromeric Instability–Facial Anomalies Syndrome Type 1

Journal of Clinical Immunology -     

Incidental Discovery of a Presacral Extradural Myxopapillary Ependymoma

Ependymoma is a malignant central nervous system tumor arising from the lining of the ventricles or central canal of the spinal cord. Extradural spinal ependymomas arise from heterotopic ependymal cells or the coccygeal medullary vestige and are extremely infrequent. We present a rare case of presacral extradural ependymoma. Extradural ependymomas typically demonstrate an extraneural spread and, thus, surveillance of the entire central nervous system is not typically recommended. A radiograph of the chest, liver profile, and attention to palpable lymphadenopathy (especially inguinal) on physical examination are vital for surveillance. Obtaining an R0 resection is the most important prognostic factor in survival and local recurrence.