Pro-angiogenic Activity Discriminates Human Adipose-Derived Stromal Cells From Retinal Pericytes: Considerations for Cell-Based Therapy of Diabetic Retinopathy

Diabetic retinopathy (DR) is a frequent diabetes-associated complication. Pericyte dropout can cause increased vascular permeability and contribute to vascular occlusion. Adipose-derived stromal cells (ASC) have been suggested to replace pericytes and restore microvascular support as potential therapy of DR. In models of DR, ASC not only generated a cytoprotective and reparative environment by the secretion of trophic factors but also engrafted and integrated into the retina in a pericyte-like fashion. The aim of this study was to compare the pro-angiogenic features of human ASC and human retinal microvascular pericytes (HRMVPC) in vitro. The proliferation and the expression of ASC and HRMVPC markers were compared. Adhesion to high glucose-conditioned endothelial extracellular matrix, mimicking the diabetic microenvironment, was measured. The angiogenesis-promoting features of both cell types and their conditioned media on human retinal endothelial cells (EC) were assessed. To identify a molecular basis for the observed differences, gene expression profiling was performed using whole-genome microarrays, and data were validated using PCR arrays and flow cytometry. Based on multiplex cytokine results, functional studies on selected growth factors were performed to assess their role in angiogenic support. Despite a distinct heterogeneity in ASC and HRMVPC cultures with an overlap of expressed markers, ASC differed functionally from HRMVPC. Most importantly, the pro-angiogenic activity was solely featured by ASC, whereas HRMVPC actively suppressed vascular network formation. HRMVPC, in contrast to ASC, showed impaired adhesion and proliferation on the high glucose-conditioned endothelial extracellular matrix. These data were supported by gene expression profiles with differentially expressed genes. The vessel-stabilizing factors were more highly expressed in HRMVPC, and the angiogenesis-promoting factors were more highly expressed in ASC. The vascular endothelial growth factor receptor-2 inhibition efficiently abolished the ASC angiogenic supportive capacities, whereas the addition of angiopoietin-1 and angiopoietin-2 did not alter these effects. Our results clearly show that ASC are pro-angiogenic, whereas HRMVPC are marked by anti-angiogenic/EC-stabilizing features. These data support ASC as pericyte replacement in DR but also suggest a careful risk-to-benefit analysis to take full advantage of the ASC therapeutic features

Pro-angiogenic Activity Discriminates Human Adipose-Derived Stromal Cells From Retinal Pericytes: Considerations for Cell-Based Therapy of Diabetic Retinopathy

Diabetic retinopathy (DR) is a frequent diabetes-associated complication. Pericyte dropout can cause increased vascular permeability and contribute to vascular occlusion. Adipose-derived stromal cells (ASC) have been suggested to replace pericytes and restore microvascular support as potential therapy of DR. In models of DR, ASC not only generated a cytoprotective and reparative environment by the secretion of trophic factors but also engrafted and integrated into the retina in a pericyte-like fashion. The aim of this study was to compare the pro-angiogenic features of human ASC and human retinal microvascular pericytes (HRMVPC) in vitro. The proliferation and the expression of ASC and HRMVPC markers were compared. Adhesion to high glucose-conditioned endothelial extracellular matrix, mimicking the diabetic microenvironment, was measured. The angiogenesis-promoting features of both cell types and their conditioned media on human retinal endothelial cells (EC) were assessed. To identify a molecular basis for the observed differences, gene expression profiling was performed using whole-genome microarrays, and data were validated using PCR arrays and flow cytometry. Based on multiplex cytokine results, functional studies on selected growth factors were performed to assess their role in angiogenic support. Despite a distinct heterogeneity in ASC and HRMVPC cultures with an overlap of expressed markers, ASC differed functionally from HRMVPC. Most importantly, the pro-angiogenic activity was solely featured by ASC, whereas HRMVPC actively suppressed vascular network formation. HRMVPC, in contrast to ASC, showed impaired adhesion and proliferation on the high glucose-conditioned endothelial extracellular matrix. These data were supported by gene expression profiles with differentially expressed genes. The vessel-stabilizing factors were more highly expressed in HRMVPC, and the angiogenesis-promoting factors were more highly expressed in ASC. The vascular endothelial growth factor receptor-2 inhibition efficiently abolished the ASC angiogenic supportive capacities, whereas the addition of angiopoietin-1 and angiopoietin-2 did not alter these effects. Our results clearly show that ASC are pro-angiogenic, whereas HRMVPC are marked by anti-angiogenic/EC-stabilizing features. These data support ASC as pericyte replacement in DR but also suggest a careful risk-to-benefit analysis to take full advantage of the ASC therapeutic features

Human carnosinase 1 overexpression aggravates diabetes and renal impairment in BTBR(Ob/Ob)mice

Objective: To assess the influence of serum carnosinase (CN1) on the course of diabetic kidney disease (DKD). Methods: hCN1 transgenic (TG) mice were generated in a BTBROb/Ob genetic background to allow the spontaneous development of DKD in the presence of serum carnosinase. The influence of serum CN1 expression on obesity, hyperglycemia, and renal impairment was assessed. We also studied if aggravation of renal impairment in hCN1 TG BTBROb/Ob mice leads to changes in the renal transcriptome as compared with wild-type BTBROb/Ob mice. Results: hCN1 was detected in the serum and urine of mice from two different hCN1 TG lines. The transgene was expressed in the liver but not in the kidney. High CN1 expression was associated with low plasma and renal carnosine concentrations, even after oral carnosine supplementation. Obese hCN1 transgenic BTBROb/Ob mice displayed significantly higher levels of glycated hemoglobin, glycosuria, proteinuria, and increased albumin-creatinine ratios (1104 ± 696 vs 492.1 ± 282.2 μg/mg) accompanied by an increased glomerular tuft area and renal corpuscle size. Gene-expression profiling of renal tissue disclosed hierarchical clustering between BTBROb/Wt, BTBROb/Ob, and hCN1 BTBROb/Ob mice. Along with aggravation of the DKD phenotype, 26 altered genes have been found in obese hCN1 transgenic mice; among them claudin-1, thrombospondin-1, nephronectin, and peroxisome proliferator–activated receptor-alpha have been reported to play essential roles in DKD. Conclusions: Our data support a role for serum carnosinase 1 in the progression of DKD. Whether this is mainly attributed to the changes in renal carnosine concentrations warrants further studies. Key messages: Increased carnosinase 1 (CN1) is associated with diabetic kidney disease (DKD).BTBROb/Ob mice with human CN1 develop a more aggravated DKD phenotype.Microarray revealed alterations by CN1 which are not altered by hyperglycemia.These genes have been described to play essential roles in DKD.Inhibiting CN1 could be beneficial in DKD

Repeated exposure to transient obstructive sleep apnea-related conditions causes an atrial fibrillation substrate in a chronic rat model

Background High night-to-night variability in obstructive sleep apnea (OSA) is associated with atrial fibrillation (AF). Obstructive apneas are characterized by intermittent deoxygenation-reoxygenation and intrathoracic pressure swings during ineffective inspiration against occluded upper airways. Objective We elucidated the effect of repeated exposure to transient OSA conditions simulated by intermittent negative upper airway pressure (INAP) on the development of an AF substrate. Methods INAP (48 events/4 h; apnea-hypopnea index 12 events/h) was applied in sedated spontaneously breathing rats (2% isoflurane) to simulate mild-to-moderate OSA. Rats without INAP served as a control group (CTR). In an acute test series (ATS), rats were either killed immediately (n = 9 per group) or after 24 hours of recovery (ATS-REC: n = 5 per group). To simulate high night-to-night variability in OSA, INAP applications (n = 10; 24 events/4 h; apnea-hypopnea index 6/h) were repeated every second day for 3 weeks in a chronic test series (CTS). Results INAP increased atrial oxidative stress acutely, represented in decreases of reduced to oxidized glutathione ratio (ATS: INAP: 0.33 ± 0.05 vs CTR: 1 ± 0.26; P = .016), which was reversible after 24 hours (ATS-REC: INAP vs CTR; P = .274). Although atrial oxidative stress did not accumulate in the CTS, atrial histological analysis revealed increased cardiomyocyte diameters, reduced connexin 43 expression, and increased interstitial fibrosis formation (CTS: INAP 7.0% ± 0.5% vs CTR 5.1% ± 0.3%; P = .013), which were associated with longer inducible AF episodes (CTS: INAP: 11.65 ± 4.43 seconds vs CTR: 0.7 ± 0.33 seconds; P = .033). Conclusion Acute simulation of OSA was associated with reversible atrial oxidative stress. Cumulative exposure to these transient OSA-related conditions resulted in AF substrates and was associated with increased AF susceptibility. Mild-to-moderate OSA with high night-to-night variability may deserve intensive management to prevent atrial substrate development

Etno-muzeji na otvorenom i zaštita spomenika kulture

Formation of the death-inducing signaling complex (DISC) initiates extrinsic apoptosis. Caspase-8 and its regulator cFLIP control death signaling by binding to death-receptor-bound FADD. By elucidating the function of the caspase-8 homolog, caspase-10, we discover that caspase-10 negatively regulates caspase-8-mediated cell death. Significantly, we reveal that caspase-10 reduces DISC association and activation of caspase-8. Furthermore, we extend our co-operative/hierarchical binding model of caspase-8/cFLIP and show that caspase-10 does not compete with caspase-8 for binding to FADD. Utilizing caspase-8-knockout cells, we demonstrate that caspase-8 is required upstream of both cFLIP and caspase-10 and that DISC formation critically depends on the scaffold function of caspase-8. We establish that caspase-10 rewires DISC signaling to NF-κB activation/cell survival and demonstrate that the catalytic activity of caspase-10, and caspase-8, is redundant in gene induction. Thus, our data are consistent with a model in which both caspase-10 and cFLIP coordinately regulate CD95L-mediated signaling for death or survival

Longitudinal transcriptome-wide gene expression analysis of sleep deprivation treatment shows involvement of circadian genes and immune pathways

Therapeutic sleep deprivation (SD) rapidly induces robust, transient antidepressant effects in a large proportion of major mood disorder patients suffering from a depressive episode, but underlying biological factors remain poorly understood. Research suggests that these patients may have altered circadian molecular genetic 'clocks' and that SD functions through 'resetting' dysregulated genes; additional factors may be involved, warranting further investigation. Leveraging advances in microarray technology enabling the transcriptome-wide assessment of gene expression, this study aimed to examine gene expression changes accompanying SD and recovery sleep in patients suffering from an episode of depression. Patients (N = 78) and controls (N = 15) underwent SD, with blood taken at the same time of day before SD, after one night of SD and after recovery sleep. A transcriptome-wide gene-by-gene approach was used, with a targeted look also taken at circadian genes. Furthermore, gene set enrichment, and longitudinal gene set analyses including the time point after recovery sleep, were conducted. Circadian genes were significantly affected by SD, with patterns suggesting that molecular clocks of responders and non-responders, as well as patients and controls respond differently to chronobiologic stimuli. Notably, gene set analyses revealed a strong widespread effect of SD on pathways involved in immune function and inflammatory response, such as those involved in cytokine and especially in interleukin signalling. Longitudinal gene set analyses showed that in responders these pathways were upregulated after SD; in non-responders, little response was observed. Our findings emphasize the close relationship between circadian, immune and sleep systems and their link to etiology of depression at the transcriptomic level

EphB2-dependent signaling promotes neuronal excitotoxicity and inflammation in the acute phase of ischemic stroke

Local cerebral hypoperfusion causes ischemic stroke while driving multiple cell-specific responses including inflammation, glutamate-induced neurotoxicity mediated via NMDAR, edema formation and angiogenesis. Despite the relevance of these pathophysiological mechanisms for disease progression and outcome, molecular determinants controlling the onset of these processes are only partially understood. In this context, our study intended to investigate the functional role of EphB2, a receptor tyrosine kinase that is crucial for synapse function and binds to membrane-associated ephrin-B ligands. Cerebral ischemia was induced in Ephb2−/− mice by transient middle cerebral artery occlusion followed by different times (6, 12, 24 and 48 h) of reperfusion. Histological, neurofunctional and transcriptome analyses indicated an increase in EphB2 phosphorylation under these conditions and attenuated progression of stroke in Ephb2−/− mice. Moreover, while infiltration of microglia/macrophages and astrocytes into the peri-infarct region was not altered, expression of the pro-inflammatory mediators MCP-1 and IL-6 was decreased in these mice. In vitro analyses indicated that binding of EphB2 to astrocytic ephrin-B ligands stimulates NF-κB-mediated cytokine expression via the MAPK pathway. Further magnetic resonance imaging of the Ephb2−/− ischemic brain revealed a lower level of cytotoxic edema formation within 6 h upon onset of reperfusion. On the mechanistic level, absence of neuronal EphB2 decreased the mitochondrial Ca2+ load upon specific activation of NMDAR but not during synaptic activity. Furthermore, neuron-specific loss of ephrin-B2 reduced the extent of cerebral tissue damage in the acute phase of ischemic stroke. Collectively, EphB2 may promote the immediate response to an ischemia-reperfusion event in the central nervous system by (i) pro-inflammatory activation of astrocytes via ephrin-B-dependent signaling and (ii) amplification of NMDA-evoked neuronal excitotoxicity

N-Octanoyl Dopamine Treatment of Endothelial Cells Induces the Unfolded Protein Response and Results in Hypometabolism and Tolerance to Hypothermia

Aim: N-acyl dopamines (NADD) are gaining attention in the field of inflammatory and neurological disorders. Due to their hydrophobicity, NADD may have access to the endoplasmic reticulum (ER). We therefore investigated if NADD induce the unfolded protein response (UPR) and if this in turn influences cell behaviour. Methods: Genome wide gene expression profiling, confirmatory qPCR and reporter assays were employed on human umbilical vein endothelial cells (HUVEC) to validate induction of UPR target genes and UPR sensor activation by N-octanoyl dopamine (NOD). Intracellular ATP, apoptosis and induction of thermotolerance were used as functional parameters to assess adaptation of HUVEC. Results: NOD, but not dopamine dose dependently induces the UPR. This was also found for other synthetic NADD. Induction of the UPR was dependent on the redox activity of NADD and was not caused by selective activation of a particular UPR sensor. UPR induction did not result in cell apoptosis, yet NOD strongly impaired cell proliferation by attenuation of cells in the S-G2/M phase. Long-term treatment of HUVEC with low NOD concentration showed decreased intracellular ATP concentration paralleled with activation of AMPK. These cells were significantly more resistant to cold inflicted injury. Conclusions: We provide for the first time evidence that NADD induce the UPR in vitro. It remains to be assessed if UPR induction is causally associated with hypometabolism and thermotolerance. Further pharmacokinetic studies are warranted to address if the NADD concentrations used in vitro can be obtained in vivo and if this in turn shows therapeutic efficacy

Methylglyoxal down-regulates the expression of cell cycle associated genes and activates the p53 pathway in human umbilical vein endothelial cells

Abstract Although methylglyoxal (MGO) has emerged as key mediator of diabetic microvascular complications, the influence of MGO on the vascular transcriptome has not thoroughly been assessed. Since diabetes is associated with low grade inflammation causing sustained nuclear factor-kappa B (NF-κB) activation, the current study addressed 1) to what extent MGO changes the transcriptome of human umbilical vein endothelial cells (HUVECs) exposed to an inflammatory milieu, 2) what are the dominant pathways by which these changes occur and 3) to what extent is this affected by carnosine, a putative scavenger of MGO. Microarray analysis revealed that exposure of HUVECs to high MGO concentrations significantly changes gene expression, characterized by prominent down-regulation of cell cycle associated genes and up-regulation of heme oxygenase-1 (HO-1). KEGG-based pathway analysis identified six significantly enriched pathways of which the p53 pathway was the most affected. No significant enrichment of inflammatory pathways was found, yet, MGO did inhibit VCAM-1 expression in Western blot analysis. Carnosine significantly counteracted MGO-mediated changes in a subset of differentially expressed genes. Collectively, our results suggest that MGO initiates distinct transcriptional changes in cell cycle/apoptosis genes, which may explain MGO toxicity at high concentrations. MGO did not augment TNF-α induced inflammation

Hypoxic Epithelial Necrosis Triggers Neutrophilic Inflammation via IL-1 Receptor Signaling in Cystic Fibrosis Lung Disease

Rationale: In many organs, hypoxic cell death triggers sterile neutrophilic inflammation via IL-1R signaling. Although hypoxia is common in airways from patients with cystic fibrosis (CF), its role in neutrophilic inflammation remains unknown. We recently demonstrated that hypoxic epithelial necrosis caused by airway mucus obstruction precedes neutrophilic inflammation in Scnn1b-transgenic (Scnn1b-Tg) mice with CF-like lung disease