Lamin A/C-dependent chromatin architecture safeguards naïve pluripotency to prevent aberrant cardiovascular cell fate and function.

Tight control of cell fate choices is crucial for normal development. Here we show that lamin A/C plays a key role in chromatin organization in embryonic stem cells (ESCs), which safeguards naïve pluripotency and ensures proper cell fate choices during cardiogenesis. We report changes in chromatin compaction and localization of cardiac genes in Lmna-/- ESCs resulting in precocious activation of a transcriptional program promoting cardiomyocyte versus endothelial cell fate. This is accompanied by premature cardiomyocyte differentiation, cell cycle withdrawal and abnormal contractility. Gata4 is activated by lamin A/C loss and Gata4 silencing or haploinsufficiency rescues the aberrant cardiovascular cell fate choices induced by lamin A/C deficiency. We uncover divergent functions of lamin A/C in naïve pluripotent stem cells and cardiomyocytes, which have distinct contributions to the transcriptional alterations of patients with LMNA-associated cardiomyopathy. We conclude that disruption of lamin A/C-dependent chromatin architecture in ESCs is a primary event in LMNA loss-of-function cardiomyopathy.

COMP-Ang-1 Improves Glucose Uptake in db/db Mice with Type 2 Diabetes.

Cartilage oligomeric matrix protein (COMP)-Angiopoietin-1 is a potent angiopoietin-1 (Ang-1) variant that possesses therapeutic potential in angiogenesis and vascular endothelial dysfunction. Noteworthy, we have shown that COMP-Ang-1 improves hyperglycemia and neuroregeneration in ob/ob mice. However, the mechanism of the antidiabetic effect of COMP-Ang-1 is completely unknown. Therefore, we elucidated the diabetes protective molecular mechanisms of COMP-Ang-1 in diabetic db/db mouse model. COMP-Ang-1 (0.5 ng/g body weight) or aqueous NaCl solution was injected intraperitoneally per day in 21 consecutive days into 3-month old, male db/db mice (n=10 per group). Blood glucose and HbA1c levels were determined at baseline and 21 days after COMP-Ang-1 or NaCl treatment. The effect of COMP-Ang-1 on glucose uptake was investigated by euglycemic-hyperinsulinemic clamp studies and key genes of glucose metabolism were studied by Western blot analysis. Our findings indicate that COMP-Ang-1 improves glucose metabolism in a tissue specific manner by regulating HIF-1α transcriptional genes of GLUT-1 expression.

The role of nerve inflammation and exogenous iron load in experimental peripheral diabetic neuropathy (PDN).

BACKGROUND: Iron is an essential but potentially toxic metal in mammals. Here we investigated a pathogenic role of exogenous iron in peripheral diabetic neuropathy (PDN) in an animal model for type 1 diabetes. METHODS: Diabetes was induced by a single injection of streptozotocin (STZ) in 4-month-old Sprague-Dawley rats. STZ-diabetic rats and non-diabetic rats were fed with high, standard, or low iron diet. After three months of feeding, animals were tested. RESULTS: STZ-rats on standard iron diet showed overt diabetes, slowed motor nerve conduction, marked degeneration of distal intraepidermal nerve fibers, mild intraneural infiltration with macrophages and T-cells in the sciatic nerve, and increased iron levels in serum and dorsal root ganglion (DRG) neurons. While motor fibers were afflicted in all STZ-groups, only a low iron-diet led also to reduced sensory conduction velocities in the sciatic nerve. In addition, only STZ-rats on a low iron diet showed damaged mitochondria in numerous DRG neurons, a more profound intraepidermal nerve fiber degeneration indicating small fiber neuropathy, and even more inflammatory cells in sciatic nerves than seen in any other experimental group. CONCLUSIONS: These results indicate that dietary iron-deficiency rather than iron overload, and mild inflammation may both promote neuropathy in STZ-induced experimental PDN.

COMP-angiopoietin-1 recovers molecular biomarkers of neuropathy and improves vascularisation in sciatic nerve of ob/ob mice.

BACKGROUND: Leptin-deficient ob/ob mice are a model of type 2 diabetes induced peripheral neuropathy. Ob/ob mice exhibit obesity, insulin resistance, hyperglycaemia, and alterations of peripheral nerve fibres and endoneural microvessels. Here we test the hypothesis that cartilage oligomeric matrix protein (COMP)-Ang-1, a soluble and stabile form of Ang-1 which promotes angiogenesis and nerve growth, improves regeneration of nerve fibres and endoneural microvessels in ob/ob mice. METHODS AND FINDINGS: COMP-Ang-1 (100 ng/ml) or NaCl were intraperitoneally (i.p.) injected into male (N = 184), 3-month old, ob/ob or ob/+ mice for 7 and 21 days. We measured expression of Nf68, GAP43, Cx32, Cx26, Cx43, and TNFα in sciatic nerves using Western blot analysis. To investigate the inflammation in sciatic nerves, numbers of macrophages and T-cells were counted after immunofluorescence staining. In ultrathin section, number of myelinated/non-mylinated nerve fibers, g-ratio, the thickness of Schwann cell basal lamina and microvessel endothelium were investigated. Endoneural microvessels were reconstructed with intracardial FITC injection. Treatment with COMP-Ang-1 over 21 days significantly reduced fasting blood glucose and plasma cholesterol concentrations compared to saline treated ob/ob mice. In addition, COMP-Ang-1 treatment: 1) up-regulated expression of Nf68 and GAP43; 2) improved expression of gap junction proteins including connexin 32 and 26; 3) suppressed the expression of TNFα and Cx43 and 4) led to decreased macrophage and T-cell infiltration in sciatic nerve of ob/ob mice. The significant changes of sciatic nerve ultrastructure were not observed after 21-day long COMP-Ang-1 treatment. COMP-Ang-1 treated ob/ob mice displayed regeneration of small-diameter endoneural microvessels. Effects of COMP-Ang-1 corresponded to increased phosphorylation of Akt and p38 MAPK upon Tie-2 receptor. CONCLUSIONS: COMP-Ang-1 recovers molecular biomarkers of neuropathy, promotes angiogenesis and suppresses inflammation in sciatic nerves of ob/ob mice suggesting COMP-Ang-1 as novel treatment option to improve morphologic and protein expression changes associated with diabetic neuropathy.

Altered sciatic nerve fiber morphology and endoneural microvessels in mouse models relevant for obesity, peripheral diabetic polyneuropathy, and the metabolic syndrome.

The morphology of sciatic nerves from leptin-deficient ob/ob mice and leptin receptor-deficient db/db mice, both models for obesity, peripheral diabetic neuropathy, and the metabolic syndrome, has yet to be examined for changes in nerve fibers and in endoneural microvessels. Sciatic nerves from three groups of 4-month-old mice (WT C57BL6, ob/ob, and db/db) were investigated. In ultrathin sections, the thickness of myelin sheaths was significantly reduced in small, medium-sized, and large axons of db/db mice compared with WT mice. In ob/ob mice, only large fibers showed a decrease in myelin sheath thickness. The number of nonmyelinated nerve fibers was lower in ob/ob mice than in the db/db group. A thickened basal lamina of Schwann cells occurred in the ob/ob group only. In contrast, the basement membrane of endoneural microvessels was thickened in both obese groups. For this reason, laminin expression in Western blot analysis was lower in the db/db group than in the ob/ob one. Endoneural microvessels, which had been injected with fluorescein isothiocyanate, depicted signs of vasodilatation in the ob/ob and vasoconstriction in db/db mice. Endoneural vessels displayed two receptors of oxLDL. LOX-1 was strongly expressed in db/db mice, whereas TLR4 was at its maximum in the ob/ob group. We conclude that changes in nerve fibers and in endoneural microvessels are present in sciatic nerve of both mouse models of type 2 diabetes. Upregulation of oxLDL-dependent receptors in endoneural microvessels might be connected to different degrees of oxidative stress in severe diabetic db/db mice and in the mild diabetic ob/ob group.

Oxidized low-density lipoprotein (oxLDL) induces cell death in neuroblastoma and survival autophagy in schwannoma cells.

Oxidized low-density lipoprotein (oxLDL) induces apoptosis or autophagy in dependence on the cell type. We here investigated the effect of oxLDL on the B104 neuroblastoma and RN22 schwannoma cells being popular in neuroscience research. Cells were cultivated with and without oxLDL. To generate oxLDL, we added 50 µg/ml nLDL and 50 µM CuSO(4) into the culture medium. After a 24-h-long treatment, oxLDL was detectable in media from both cell culture types and its concentration was approximately 16 µg/ml. In the oxLDL-treated B104 neuroblastoma cell cultures 75% cells died after the 24-h exposure. The intact cells showed impaired mitochondria at the ultrastructural level. Western blot analysis revealed the increased expression of AIF 57 kDa (AIF(57)) protein, as a sign of caspase-independent cell death. In RN22 schwannoma cell cultures, oxLDL did not have any effect on cleaved caspase-3 and AIF(57) protein levels indicating absence of cell death. Treated RN22 schwannoma cells underwent survival autophagy by forming conspicuous autophagosomes and by processing LC3-I into LC3-II protein. Collectively, oxLDL induces AIF-dependent cell death in B104 neuroblastoma cells whereas in RN22 schwannoma cells enhanced signs of survival autophagy are noted.

Oxidized low-density lipoprotein (oxLDL)-induced cell death in dorsal root ganglion cell cultures depends not on the lectin-like oxLDL receptor-1 but on the toll-like receptor-4.

DRG cells have been found to undergo apoptosis and necrosis after oxidized low-density lipoprotein (oxLDL) stimulation in vitro. However, the mechanism of oxLDL-induced DRG cell death is unclear. For this reason, we studied the expression of two potential oxLDL receptors: lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and toll-like receptor-4 (TLR4) in dorsal root ganglion (DRG) cell cultures from postnatal rats. Cells were cultivated with and without oxLDL. In oxLDL-treated DRG cell cultures, the increase of cleaved caspase-3 protein was observed as a sign of enhanced apoptosis. Untreated and oxLDL-treated DRG cell cultures expressed LOX-1 and TLR4 at similar levels. The LOX-1 expression remained unchanged after receptor blockade. However, the inhibition of LOX-1 caused a significant increase of cleaved caspase-3 and a decrease of TLR4 levels. The TLR4-inhibited DRG cell cultures lacked changes in LOX-1 expression for all experimental groups. The inhibition of TLR4 caused activation of jun N-terminal kinase (JNK) and a significant decrease of cleaved caspase-3 but did not change the TLR4 level. We conclude that LOX-1 and TLR4 are expressed in cultivated rat DRG cells and that the oxLDL-induced cell death in DRG cell cultures does not depend on the LOX-1 but on the TLR4.

Elevated levels of oxidized low-density lipoprotein and of catalase activity in follicular fluid of obese women.

The intrafollicular levels of oxidized low-density lipoprotein (oxLDL) and of enzyme antioxidants might contribute to reproductive disorders in obese and infertile women. Relevant data are missing. Eighty-four patients were grouped according to obese versus non-obese status and whether they had polycystic ovary syndrome (PCOS). The concentrations of oxLDL and the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione reductase (GR) in the serum and follicular fluid were measured. Obese women with and without PCOS had significantly greater amounts of oxLDL in the follicular fluid as compared with non-obese women. The level of oxLDL in the follicular fluid was 1000 times lower than in serum. Obese women with and without PCOS had significantly higher catalase activity in the follicular fluid as compared with non-obese women. No differences were found for the SOD activity in the follicular fluid. The GPx and GR activities were up-regulated in obese patients without and with PCOS, yet not in respect to each serum and follicular fluid sample. We conclude that elevated levels of oxLDL in the follicular fluid of obese women are associated with higher catalase activity; both parameters are independent of PCOS. The levels of oxLDL and catalase activity appear to indicate different degrees of oxidative stress.

Granulosa cell subtypes respond by autophagy or cell death to oxLDL-dependent activation of the oxidized lipoprotein receptor 1 and toll-like 4 receptor.

Autophagic cell death has been observed in granulosa cell cultures via the oxLDL-dependent activation of lectin-like oxidized low density lipoprotein receptor 1 (LOX-1). This activation might differ for cytokeratin-positive (CK(+)) and CK(-) granulosa cells. In particular, LOX-1 and toll-like receptor 4 (TLR4), one of the pattern recognition receptors of innate immunity, might be diversely regulated. Granulosa cell subtype cultures were established from the follicle harvests of patients undergoing in vitro fertilization (IVF) therapy. In response to oxLDL treatment, the fibroblast-like CK(-) cells upregulated LOX-1 and exhibited reparative autophagy, which could be blocked with anti-LOX-1 antibody. The epithelioid-like CK(+) cells did not regulate LOX-1 expression upon oxLDL application, but the expression of TLR4 and CD14 increased between 0 and 36 h of oxLDL/nDL treatment. This upregulation was associated with nonapoptotic cell death based on the absence of cleaved caspase-3. Reactive oxygen species (ROS) increased with 12 h oxLDL application and steroidogenic acute regulatory (StAR) protein expression was negligible. In CK(-) cells, the inhibition of TLR4 downregulated LOX-1 and induced apoptosis. We concluded that CK(-) granulosa cells are protected against oxLDL-dependent apoptosis by TLR4, whereas, in CK(+) cells, oxLDL-induced TLR4 activation triggers nonapoptotic cell death. The CK(+) cells might represent immune-like granulosa cells involved in ovarian remodeling processes.