Madalina Opreanu

Diabetic retinopathy is associated with bone marrow neuropathy and a depressed peripheral clock

The present epidemic of diabetes is resulting in a worldwide increase in cardiovascular and microvascular complications including retinopathy. Current thinking has focused on local influences in the retina as being responsible for development of this diabetic complication. However, the contribution of circulating cells in maintenance, repair, and dysfunction of the vasculature is now becoming appreciated. Diabetic individuals have fewer endothelial progenitor cells (EPCs) in their circulation and these cells have diminished migratory potential, which contributes to their decreased reparative capacity. Using a rat model of type 2 diabetes, we show that the decrease in EPC release from diabetic bone marrow is caused by bone marrow neuropathy and that these changes precede the development of diabetic retinopathy. In rats that had diabetes for 4 mo, we observed a dramatic reduction in the number of nerve terminal endings in the bone marrow. Denervation was accompanied by increased numbers of EPCs within the bone marrow but decreased numbers in circulation. Furthermore, denervation was accompanied by a loss of circadian release of EPCs and a marked reduction in clock gene expression in the retina and in EPCs themselves. This reduction in the circadian peak of EPC release led to diminished reparative capacity, resulting in the development of the hallmark feature of diabetic retinopathy, acellular retinal capillaries. Thus, for the first time, diabetic retinopathy is related to neuropathy of the bone marrow. This novel finding shows that bone marrow denervation represents a new therapeutic target for treatment of diabetic vascular complications.

Remodeling of Retinal Fatty Acids in an Animal Model of Diabetes: A Decrease in Long-Chain Polyunsaturated Fatty Acids Is Associated With a Decrease in Fatty Acid Elongases Elovl2 and Elovl4

OBJECTIVE The results of the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications cohort study revealed a strong association between dyslipidemia and the development of diabetic retinopathy. However, there are no experimental data on retinal fatty acid metabolism in diabetes. This study determined retinal-specific fatty acid metabolism in control and diabetic animals. RESEARCH DESIGN AND METHODS Tissue gene and protein expression profiles were determined by quantitative RT-PCR and Western blot in control and streptozotocin-induced diabetic rats at 3–6 weeks of diabetes. Fatty acid profiles were assessed by reverse-phase high-performance liquid chromatography, and phospholipid analysis was performed by nano-electrospray ionization tandem mass spectrometry. RESULTS We found a dramatic difference between retinal and liver elongase and desaturase profiles with high elongase and low desaturase gene expression in the retina compared with liver. Elovl4, an elongase expressed in the retina but not in the liver, showed the greatest expression level among retinal elongases, followed by Elovl2, Elovl1, and Elovl6. Importantly, early-stage diabetes induced a marked decrease in retinal expression levels of Elovl4, Elovl2, and Elovl6. Diabetes-induced downregulation of retinal elongases translated into a significant decrease in total retinal docosahexaenoic acid, as well as decreased incorporation of very-long-chain polyunsaturated fatty acids (PUFAs), particularly 32:6n3, into retinal phosphatidylcholine. This decrease in n3 PUFAs was coupled with inflammatory status in diabetic retina, reflected by an increase in gene expression of proinflammatory markers interleukin-6, vascular endothelial growth factor, and intercellular adhesion molecule-1. CONCLUSIONS This is the first comprehensive study demonstrating diabetes-induced changes in retinal fatty acid metabolism. Normalization of retinal fatty acid levels by dietary means or/and modulating expression of elongases could represent a potential therapeutic target for diabetes-induced retinal inflammation.

The Unconventional Role of Acid Sphingomyelinase in Regulation of Retinal Microangiopathy in Diabetic Human and Animal Models

OBJECTIVE Acid sphingomyelinase (ASM) is an important early responder in inflammatory cytokine signaling. The role of ASM in retinal vascular inflammation and vessel loss associated with diabetic retinopathy is not known and represents the goal of this study. RESEARCH DESIGN AND METHODS Protein and gene expression profiles were determined by quantitative RT-PCR and Western blot. ASM activity was determined using Amplex Red sphingomyelinase assay. Caveolar lipid composition was analyzed by nano-electrospray ionization tandem mass spectrometry. Streptozotocin-induced diabetes and retinal ischemia-reperfusion models were used in in vivo studies. RESULTS We identify endothelial caveolae-associated ASM as an essential component in mediating inflammation and vascular pathology in in vivo and in vitro models of diabetic retinopathy. Human retinal endothelial cells (HREC), in contrast with glial and epithelial cells, express the plasma membrane form of ASM that overlaps with caveolin-1. Treatment of HREC with docosahexaenoic acid (DHA) specifically reduces expression of the caveolae-associated ASM, prevents a tumor necrosis factor-α–induced increase in the ceramide-to-sphingomyelin ratio in the caveolae, and inhibits cytokine-induced inflammatory signaling. ASM is expressed in both vascular and neuroretina; however, only vascular ASM is specifically increased in the retinas of animal models at the vasodegenerative phase of diabetic retinopathy. The absence of ASM in ASM−/− mice or inhibition of ASM activity by DHA prevents acellular capillary formation. CONCLUSIONS This is the first study demonstrating activation of ASM in the retinal vasculature of diabetic retinopathy animal models. Inhibition of ASM could be further explored as a potential therapeutic strategy in treating diabetic retinopathy.

N-3 Polyunsaturated Fatty Acids Prevent Diabetic Retinopathy by Inhibition of Retinal Vascular Damage and Enhanced Endothelial Progenitor Cell Reparative Function

Objective The vasodegenerative phase of diabetic retinopathy is characterized by not only retinal vascular degeneration but also inadequate vascular repair due to compromised bone marrow derived endothelial progenitor cells (EPCs). We propose that n-3 polyunsaturated fatty acid (PUFA) deficiency in diabetes results in activation of the central enzyme of sphingolipid metabolism, acid sphingomyelinase (ASM) and that ASM represents a molecular metabolic link connecting the initial damage in the retina and the dysfunction of EPCs. Research Design and Methods Type 2 diabetic rats on control or docosahexaenoic acid (DHA)-rich diet were studied. The number of acellular capillaries in the retinas was assessed by trypsin digest. mRNA levels of interleukin (IL)-1β, IL-6, intracellular adhesion molecule (ICAM)-1 in the retinas from diabetic animals were compared to controls and ASM protein was assessed by western analysis. EPCs were isolated from blood and bone marrow and their numbers and ability to form colonies in vitro, ASM activity and lipid profiles were determined. Results DHA-rich diet prevented diabetes-induced increase in the number of retinal acellular capillaries and significantly enhanced the life span of type 2 diabetic animals. DHA-rich diet blocked upregulation of ASM and other inflammatory markers in diabetic retina and prevented the increase in ASM activity in EPCs, normalized the numbers of circulating EPCs and improved EPC colony formation. Conclusions In a type 2 diabetes animal model, DHA-rich diet fully prevented retinal vascular pathology through inhibition of ASM in both retina and EPCs, leading to a concomitant suppression of retinal inflammation and correction of EPC number and function.

Inhibition of cytokine signaling in human retinal endothelial cells through downregulation of sphingomyelinases by docosahexaenoic acid.

PURPOSE The authors have previously demonstrated that DHA inhibits cytokine-induced inflammation in human retinal endothelial cells (HRECs), the resident vasculature affected by diabetic retinopathy. However, the anti-inflammatory mechanism of docosahexaenoic acid (DHA) is still not well understood. Sphingolipids represent a major component of membrane microdomains, and ceramide-enriched microdomains appear to be a prerequisite for inflammatory cytokine signaling. Acid sphingomyelinase (ASMase) and neutral sphingomyelinase (NSMase) are key regulatory enzymes of sphingolipid metabolism, promoting sphingomyelin hydrolysis to proinflammatory ceramide. The authors address the hypothesis that DHA inhibits cytokine-induced inflammatory signaling in HRECs by downregulating sphingomyelinases. METHODS ASMase and NSMase activity was determined by sphingomyelinase assay in primary cultures of HRECs. The expression of ASMase, NSMase, ICAM-1, and VCAM-1 was assessed by quantitative PCR and Western blot analysis. Gene silencing of ASMase and NSMase was obtained by siRNA treatment. RESULTS Inflammatory cytokines TNFalpha and IL-1beta induced cellular adhesion molecule (CAM) expression and rapid increase in ASMase and NSMase activity in HRECs. DHA decreased basal and cytokine-induced ASMase and NSMase expression and activity and the upregulation of CAM expression. Anti-inflammatory effects of DHA on cytokine-induced CAM expression were mimicked by inhibition/gene silencing of ASMase and NSMase. The sphingomyelinase pathway rather than ceramide de novo synthesis pathway was important for inflammatory signaling in HRECs. CONCLUSIONS This study provides a novel potential mechanism for the anti-inflammatory effect of DHA in HRECs. DHA downregulates the basal and cytokine-induced ASMase and NSMase expression and activity level in HRECs, and inhibition of sphingomyelinases in endothelial cells prevents cytokine-induced inflammatory response.

Wearable cardioverter-defibrillator as a bridge to cardiac transplantation: A national database analysis

BACKGROUND Life-threatening ventricular arrhythmias (VAs) and sudden cardiac death (SCD) are common in patients awaiting heart transplantation (HT), and the implantable cardioverter-defibrillator (ICD) is often used for primary prevention in this setting. Use of ICDs in these patients is not without risks and is sometimes contraindicated. The wearable cardioverter-defibrillator (WCD) may be a reasonable alternative to bridge the period of risk leading up to HT. METHODS We obtained a convenience sample of patients prescribed an WCD as a bridge therapy to HT. The available data consisted of demographics, cardiac transplantation status, associated comorbidities, device use, device-stored electrocardiogram (ECG) and reason for discontinuing the WCD. Statistical analyses were performed using SPSS version 17 and GraphPad PRISM 5. RESULTS The registry included 121 patients consisting of 83 (69%) men and 38 (31%) women. The mean age was 44 ± 18 years. Mean ejection fraction was 25 ± 15%. Non-ischemic cardiomyopathy (CMP) was the underlying diagnosis in 67 (55%) patients, whereas 21 (17%) patients had ischemic CMP and 33 (27%) had a mixed or uncharacterized CMP. New York Heart Association Class III heart failure was present in 32% and 34% were in Class IV. Eighty-eight patients (73%) were being evaluated for HT or were on an HT waiting list, and 33 patients (27%) had had a prior HT, experienced rejection, and were awaiting re-transplantation. The patients wore the WCD for an average of 127 ± 392 days (median 39 days) with average daily use of 17 ± 7 hours (median 20 hours). Seven patients (6%) received appropriate WCD shocks. Fifty-one patients (42%) ended use after ICD implantation and 13 patients (11%) after HT. There were 11 deaths (9%). CONCLUSIONS A significant proportion of patients on the HT waiting list will have VA. WCD use in our study showed high compliance and efficacy and a low complication rate, suggesting that the WCD is a reasonable bridge therapy for preventing SCD in patients awaiting HT.

National Experience with Wearable Cardioverter-Defibrillator Use in Takotsubo Cardiomyopathy

Takotsubo cardiomyopathy (TCM) is generally a reversible cardiomyopathy with a favorable prognosis. Because of a risk of sudden cardiac death (SCD), a wearable cardioverter- defibrillator (WCD) is occasionally prescribed, although its utility is unknown. We reviewed a national database of TCM patients who were prescribed a WCD. The database collected baseline demographics, left ventricular ejection fraction (EF), usage compliance, documented arrhythmias, and final survival status. One-hundred and two patients with mean age 63i12 years, 11% men, had an initial EF of 27i7% at the time of WCD prescription. The mean days of use was 44i31 days, with an average daily hours used of 20i4 hours. The average follow-up period was 440i374 days and 95% of patients wore the WCD .90% of prescribed days. Two patients (2%) experienced shocks for ventricular arrhythmias (VAs) and survived; two patients (2%) experienced significant bradyarrhythmias; one patient received two inappropriate shocks due to signal artifact; no patients experienced a detection failure; two patients died during the prescription period: one with asystole, and one while not wearing the WCD; five patients died after discontinuing WCD usage, two of whom had an EF §35% at the time of WCD discontinuation. The WCD was used with a compliance of .90%. The device detected VAs reliably with a low risk of inappropriate shocks. TCM may be associated with a significant risk of death due to tachy- or bradyarrhythmias and the risk of SCD may persist even if the EF improves.