Imbalances in Mobilization and Activation of Pro-Inflammatory and Vascular Reparative Bone Marrow-Derived Cells in Diabetic Retinopathy

Diabetic retinopathy is a sight-threatening complication of diabetes, affecting 65% of patients after 10 years of the disease. Diabetic metabolic insult leads to chronic low-grade inflammation, retinal endothelial cell loss and inadequate vascular repair. This is partly due to bone marrow (BM) pathology leading to increased activity of BM-derived pro-inflammatory monocytes and impaired function of BM-derived reparative circulating angiogenic cells (CACs). We propose that diabetes has a significant long-term effect on the nature and proportion of BM-derived cells that circulate in the blood, localize to the retina and home back to their BM niche. Using a streptozotocin mouse model of diabetic retinopathy with GFP BM-transplantation, we have demonstrated that BM-derived circulating pro-inflammatory monocytes are increased in diabetes while reparative CACs are trapped in the BM and spleen, with impaired release into circulation. Diabetes also alters activation of splenocytes and BM-derived dendritic cells in response to LPS stimulation. A majority of the BM-derived GFP cells that migrate to the retina express microglial markers, while others express endothelial, pericyte and Müller cell markers. Diabetes significantly increases infiltration of BM-derived microglia in an activated state, while reducing infiltration of BM-derived endothelial progenitor cells in the retina. Further, control CACs injected into the vitreous are very efficient at migrating back to their BM niche, whereas diabetic CACs have lost this ability, indicating that the in vivo homing efficiency of diabetic CACs is dramatically decreased. Moreover, diabetes causes a significant reduction in expression of specific integrins regulating CAC migration. Collectively, these findings indicate that BM pathology in diabetes could play a role in both increased pro-inflammatory state and inadequate vascular repair contributing to diabetic retinopathy

Increase in acid sphingomyelinase level in human retinal endothelial cells and CD34+ circulating angiogenic cells isolated from diabetic individuals is associated with dysfunctional retinal vasculature and vascular repair process in diabetes

BACKGROUND: Diabetic retinopathy is a microvascular disease that results from retinal vascular degeneration and defective repair due to diabetes-induced endothelial progenitor dysfunction. OBJECTIVE: Understanding key molecular factors involved in vascular degeneration and repair is paramount for developing effective diabetic retinopathy treatment strategies. We propose that diabetes-induced activation of acid sphingomyelinase (ASM) plays essential role in retinal endothelial and CD34+ circulating angiogenic cell (CAC) dysfunction in diabetes. METHODS: Human retinal endothelial cells (HRECs) isolated from control and diabetic donor tissue and human CD34+ CACs from control and diabetic patients were used in this study. ASM messenger RNA and protein expression were assessed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. To evaluate the effect of diabetes-induced ASM on HRECs and CD34+ CACs function, tube formation, CAC incorporation into endothelial tubes, and diurnal release of CD34+ CACs in diabetic individuals were determined. RESULTS: ASM expression level was significantly increased in HRECs isolated from diabetic compared with control donor tissue, as well as CD34+ CACs and plasma of diabetic patients. A significant decrease in tube area was observed in HRECs from diabetic donors compared with control HRECs. The tube formation deficiency was associated with increased expression of ASM in diabetic HRECs. Moreover, diabetic CD34+ CACs with high ASM showed defective incorporation into endothelial tubes. Diurnal release of CD34+ CACs was disrupted with the rhythmicity lost in diabetic patients. CONCLUSION: Collectively, these findings support that diabetes-induced ASM upregulation has a marked detrimental effect on both retinal endothelial cells and CACs

Role of Acid Sphingomyelinase in Shifting the Balance Between Proinflammatory and Reparative Bone Marrow Cells in Diabetic Retinopathy

The metabolic insults associated with diabetes lead to low-grade chronic inflammation, retinal endothelial cell damage, and inadequate vascular repair. This is partly due to the increased activation of bone marrow (BM)-derived proinflammatory monocytes infiltrating the retina, and the compromised function of BM-derived reparative circulating angiogenic cells (CACs), which home to sites of endothelial injury and foster vascular repair. We now propose that a metabolic link leading to activated monocytes and dysfunctional CACs in diabetes involves upregulation of a central enzyme of sphingolipid signaling, acid sphingomyelinase (ASM). Selective inhibition of ASM in the BM prevented diabetes-induced activation of BM-derived microglia-like cells and normalized proinflammatory cytokine levels in the retina. ASM upregulation in diabetic CACs caused accumulation of ceramide on their cell membrane, thereby reducing membrane fluidity and impairing CAC migration. Replacing sphingomyelin with ceramide in synthetic membrane vesicles caused a similar decrease in membrane fluidity. Inhibition of ASM in diabetic CACs improved membrane fluidity and homing of these cells to damaged retinal vessels. Collectively, these findings indicate that selective modulation of sphingolipid metabolism in BM-derived cell populations in diabetes normalizes the reparative/proinflammatory cell balance and can be explored as a novel therapeutic strategy for treating diabetic retinopathy

Dual Anti-Inflammatory and Anti-Angiogenic Action of miR-15a in Diabetic Retinopathy

AbstractActivation of pro-inflammatory and pro-angiogenic pathways in the retina and the bone marrow contributes to pathogenesis of diabetic retinopathy. We identified miR-15a as key regulator of both pro-inflammatory and pro-angiogenic pathways through direct binding and inhibition of the central enzyme in the sphingolipid metabolism, ASM, and the pro-angiogenic growth factor, VEGF-A. miR-15a was downregulated in diabetic retina and bone marrow cells. Over-expression of miR-15a downregulated, and inhibition of miR-15a upregulated ASM and VEGF-A expression in retinal cells. In addition to retinal effects, migration and retinal vascular repair function was impaired in miR-15a inhibitor-treated circulating angiogenic cells (CAC). Diabetic mice overexpressing miR-15a under Tie-2 promoter had normalized retinal permeability compared to wild type littermates. Importantly, miR-15a overexpression led to modulation toward nondiabetic levels, rather than complete inhibition of ASM and VEGF-A providing therapeutic effect without detrimental consequences of ASM and VEGF-A deficiencies

Somatotopic principle of perineural implantation of stem cells in patients with brain injuries

peer reviewedBackground: Neuro destructive processes of any etiology are related to problematic and socially important diseases due to ineffective therapeutic strategy and need to search for new successful ways of treatment and rehabilitation of patient with cerebral infarctions and brain attacks Aims: Authors plant overify hypothesis on viability of additional use of perineural implantation of autologous mesenchymal stem cells (MSC) in order to optimize standard therapy of patients with brain attacks. Such combined technology is aim datextra activation of brain plasticity mechanisms during development of neuro destructive processes. Methods: The technique of MSC perineural migration to injured brain regions was experimentally verifed on rats (n=40) paying attention to somatotopic organization of cranial nerves. This technique was clinically tested in pilot project. Phenotyping of autologous MSC from adipose tissue (AT) was performed in 23 patients with brain attacks. These 23 patients received standard treatment as per international guidelines together with three perineural implantations of autologous MSC from AT with 5-9days intervals. The other group of patients (n=7) received only standard therapy as per international guidelines. Results: Additional use of cell therapy resulted in more rapid and effective recovery of disordered neurological functions in all cases compared to those who received standard therapy. The phenomenon of abrupt recovery of neurological functions was established during frst 24hours after each injection of autologous MSC. Cumulative recovery of functions progressed after each implantation. Discussion and conclusion: Experimentally developed technique of perineural implantation of autologous MSC was successfully verifed in clinical conditions in accordance with certifed cell therapy guideline (The Ministry of Health of the Republic of Belarus) in combination with standard treatment of patients with cerebral infarctions. Cell therapy with autologous MSC from AT by means of perineural delivery to injured brain regions is the basis for activation of reparative potential of nerve tissue and progressive recovery of neurological functions in patients with cerebral infarctions

Case Study of Selected Network Vulnerabilities

The main goal of this thesis is to deal with databases of vulnerable code bases and vulnerable applications, and to implement a tool for autonomous search and saving data from those databases to a local one. The thesis is divided into theoretical and practical parts. The theoretical part deals with my current knowledge of the main topic and creates a foundation for the implementation. Various kinds of vulnerabilities and network attacks are described in detail in this part. The practical part describes implementation of the tool and its real use

Diabetes reduces number of BM-derived endothelial cells in retina.

<p>GFP⁺ endothelial cells (gated as CD45^- Tie-2⁺ CD31⁺ cells) are expressed as a percentage of total GFP⁺ retinal cells, and indicate a significant decrease in BM-derived endothelial cells in diabetic mice. Representative flow charts of GFP⁺ endothelial cells in the retina are shown. N = 4–5.</p

Diabetes alters BM-derived microglia in retina.

<p>(A) Percentage of BM-derived microglia per mm² area of control or diabetic retina. N = 7–8. (B) GFP⁺ microglia (gated as GFP⁺ cells that are Thy1^-, Ly6G^-, Ly6C^-, CD45^dim CD11b⁺ cells) are expressed as a percentage of total GFP⁺ retinal cells in control and diabetic retina of chimeric mice, and indicate increase in BM-derived microglia in diabetic mice. Representative flow charts of GFP⁺ microglia in the retina are shown. N = 3–5, ** p< 0.01. (C) Confocal images of retina isolated from control or diabetic GFP⁺ BM-transplanted mice. Microglial marker Iba-1⁺ staining (red) with GFP⁺ (green) cells, showing colocalization (yellow) in retina. Increased retraction of processes observed in BM-derived microglia in diabetic GFP⁺ chimeric mouse retina compared to ramified, resting phenotype of microglia in control retinas (white arrowheads). Scale bars are 50 μm. (D) Quantification of dendrite length of microglia in diabetic and control chimeric mouse retinas is shown. N = 4–5, *** p< 0.001.</p

Diabetes alters response of BM-derived cells and splenocytes to LPS stimulation.

<p>Increased secretion of cytokines IL-1β and TNF-α in (A) BM-derived dendritic cell-enriched population (B) splenocytes stimulated with LPS. N = 4–5, * p< 0.05.</p

Diabetes alters release of vascular reparative cells into circulation.

<p>(A) CACs as a percentage of total bone marrow cells, *** p<0.001 (B) CACs as a percentage of total splenocytes. (C) CACs as a percentage of total blood cells. CACs are gated as Lin^- CD34⁺ CD309⁺ cells. Representative flow charts of GFP⁺ CACs in control and diabetic BM, spleen and blood are shown below. * p<0.05, N = 4–8.</p