A time window for rescuing dying retinal ganglion cells.

BACKGROUND: Retinal ganglion cell (RGC) degeneration and death cause vision loss in patients with glaucoma. Regulated cell death, once initiated, is generally considered to be an irreversible process. Recently, we showed that, by timely removing the cell death stimulus, stressed neuronal PC12 cells can recover from phosphatidylserine (PS) exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation, mitochondrial membrane potential loss, and retraction of neurites, all hallmarks of an activated cell death program. Whether the cell death process can be reversed in neurons of the central nervous system, like RGCs, is still unknown. Here, we studied reversibility of the activated cell death program in primary rat RGCs (prRGCs). METHODS: prRGCs were exposed to ethanol (5%, vol/vol) to induce cell death. At different stages of the cell death process, ethanol was removed by washing and injured prRGCs were further cultured in fresh medium to see whether they recovered. The dynamics of single cells were monitored by high-resolution live-cell spinning disk microscopy. PS exposure, mitochondrial structure, membrane potential, and intracellular Ca2+ were revealed by annexin A5-FITC, Mito-tracker, TMRM, and Fluo 8-AM staining, respectively. The distribution of cytochrome c was investigated by immunofluorescence. The ultrastructure of mitochondria was studied by electron microscopy. RESULTS: Analysis of temporal relationships between mitochondrial changes and PS exposure showed that fragmentation of the mitochondrial network and loss of mitochondrial membrane potential occurred before PS exposure. Mitochondrial changes proceeded caspase-independently, while PS exposure was caspase dependent. Interestingly, prRGCs recovered quickly from these mitochondrial changes but not from PS exposure at the plasma membrane. Correlative light and electron microscopy showed that stress-induced decrease in mitochondrial area, length and cristae number was reversible. Intracellular Ca2+ was elevated during this stage of reversible mitochondrial injury, but there was no sign of mitochondrial cytochrome c release. CONCLUSIONS: Our study demonstrates that RGCs with impaired mitochondrial structure and function can fully recover if there is no mitochondrial cytochrome c release yet, and no PS is exposed at the plasma membrane. This finding indicates that there is a time window for rescuing dying or injured RGCs, by simply removing the cell death stimulus. Video Abstract.

M6229 Protects against Extracellular-Histone-Induced Liver Injury, Kidney Dysfunction, and Mortality in a Rat Model of Acute Hyperinflammation.

Extracellular histones have been shown to act as DAMPs in a variety of inflammatory diseases. Moreover, they have the ability to induce cell death. In this study, we show that M6229, a low-anticoagulant fraction of unfractionated heparin (UFH), rescues rats that were challenged by continuous infusion of calf thymus histones at a rate of 25 mg histones/kg/h. Histone infusion by itself induced hepatic and homeostatic dysfunction characterized by elevated activity of hepatic enzymes (ASAT and ALAT) and serum lactate levels as well as by a renal dysfunction, which contributed to the significantly increased mortality rate. M6229 was able to restore normal levels of both hepatic and renal parameters at 3 and 9 mg M6229/kg/h and prevented mortality of the animals. We conclude that M6229 is a promising therapeutic agent to treat histone-mediated disease.

Assessment of longitudinal serum neutrophil extracellular trap-inducing activity in anti-neutrophil cytoplasmic antibody-associated vasculitis and glomerulonephritis in a prospective cohort using a novel bio-impedance technique.

Neutrophil extracellular traps (NET) have been implicated in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Here, we developed a novel, label-free, high-throughput bio-impedance technique to effectively measure serum NET-inducing activity. Using this technique, NET-inducing activity of serum derived from patients with AAV was assessed in a prospective cohort of 62 patients presenting with active AAV with major organ involvement. Thirty-five patients presented with new and 27 patients presented with relapsing AAV, of whom 38 had kidney and/or 31 had lung involvement. NET-inducing activity was assessed at diagnosis of active AAV (time zero), during the first 6 weeks of treatment, and after 6 months of treatment. Forty-seven patients revealed elevated NET-inducing activity at time zero. After initiation of immunosuppressive treatment, NET-inducing activity was reduced at six weeks. A subsequent increase at six months could potentially identify patients with relapsing disease (hazard ratio, 11.45 [interquartile range 1.36-96.74]). NET-inducing activity at time zero correlated with kidney function and proteinuria. Importantly, in kidney tissue, NETs co-localized with lesions typical of ANCA-associated glomerulonephritis and even correlated with systemic serum NET-inducing activity. Thus, our prospective data corroborate the importance of NET formation in AAV and ANCA-associated glomerulonephritis and the potential of longitudinal evaluation, as monitored by our novel bio-impedance assay and detailed histological evaluation.

Inhibition of Neutral Sphingomyelinase 2 by Novel Small Molecule Inhibitors Results in Decreased Release of Extracellular Vesicles by Vascular Smooth Muscle Cells and Attenuated Calcification.

Vascular calcification (VC) is an important contributor and prognostic factor in the pathogenesis of cardiovascular diseases. VC is an active process mediated by the release of extracellular vesicles by vascular smooth muscle cells (VSMCs), and the enzyme neutral sphingomyelinase 2 (nSMase2 or SMPD3) plays a key role. Upon activation, the enzyme catalyzes the hydrolysis of sphingomyelin, thereby generating ceramide and phosphocholine. This conversion mediates the release of exosomes, a type of extracellular vesicles (EVs), which ultimately forms the nidus for VC. nSMase2 therefore represents a drug target, the inhibition of which is thought to prevent or halt VC progression. In search of novel druglike small molecule inhibitors of nSMase2, we have used virtual ligand screening to identify potential ligands. From an in-silico collection of 48,6844 small druglike molecules, we selected 996 compounds after application of an in-house multi-step procedure combining different filtering and docking procedures. Selected compounds were functionally tested in vitro; from this, we identified 52 individual hit molecules that inhibited nSMase2 activity by more than 20% at a concentration of 150 µM. Further analysis showed that five compounds presented with IC50s lower than 2 µM. Of these, compounds ID 5728450 and ID 4011505 decreased human primary VSMC EV release and calcification in vitro. The hit molecules identified here represent new classes of nSMase2 inhibitors that may be developed into lead molecules for the therapeutic or prophylactic treatment of VC.

Time Dependent Changes in the Ovine Neurovascular Unit; A Potential Neuroprotective Role of Annexin A1 in Neonatal Hypoxic-Ischemic Encephalopathy.

Perinatal brain injury following hypoxia-ischemia (HI) is characterized by high mortality rates and long-term disabilities. Previously, we demonstrated that depletion of Annexin A1, an essential mediator in BBB integrity, was associated with a temporal loss of blood-brain barrier (BBB) integrity after HI. Since the molecular and cellular mechanisms mediating the impact of HI are not fully scrutinized, we aimed to gain mechanistic insight into the dynamics of essential BBB structures following global HI in relation to ANXA1 expression. Global HI was induced in instrumented preterm ovine fetuses by transient umbilical cord occlusion (UCO) or sham occlusion (control). BBB structures were assessed at 1, 3, or 7 days post-UCO by immunohistochemical analyses of ANXA1, laminin, collagen type IV, and PDGFRß for pericytes. Our study revealed that within 24 h after HI, cerebrovascular ANXA1 was depleted, which was followed by depletion of laminin and collagen type IV 3 days after HI. Seven days post-HI, increased pericyte coverage, laminin and collagen type IV expression were detected, indicating vascular remodeling. Our data demonstrate novel mechanistic insights into the loss of BBB integrity after HI, and effective strategies to restore BBB integrity should potentially be applied within 48 h after HI. ANXA1 has great therapeutic potential to target HI-driven brain injury.

Conformational plasticity of ADAMTS13 in hemostasis and autoimmunity.

A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) is a multidomain metalloprotease for which until now only a single substrate has been identified. ADAMTS13 cleaves the polymeric force-sensor von Willebrand factor (VWF) that unfolds under shear stress and recruits platelets to sites of vascular injury. Shear force-dependent cleavage at a single Tyr-Met peptide bond in the unfolded VWF A2 domain serves to reduce the size of VWF polymers in circulation. In patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP), a rare life-threatening disease, ADAMTS13 is targeted by autoantibodies that inhibit its activity or promote its clearance. In the absence of ADAMTS13, VWF polymers are not adequately processed, resulting in spontaneous adhesion of blood platelets, which presents as severe, life-threatening microvascular thrombosis. In healthy individuals, ADAMTS13-VWF interactions are guided by controlled conversion of ADAMTS13 from a closed, inactive to an open, active conformation through a series of interdomain contacts that are now beginning to be defined. Recently, it has been shown that ADAMTS13 adopts an open conformation in the acute phase and during subclinical disease in iTTP patients, making open ADAMTS13 a novel biomarker for iTTP. In this review, we summarize our current knowledge on ADAMTS13 conformation and speculate on potential triggers inducing conformational changes of ADAMTS13 and how these relate to the pathogenesis of iTTP.

Single Cell Analysis of Reversibility of the Cell Death Program in Ethanol-Treated Neuronal PC12 Cells.

Neurodegenerative diseases are generally characterized clinically by the selective loss of a distinct subset of neurons and a slow progressive course. Mounting evidence in vivo indicates that large numbers of neurons pass through a long period of injury and dysfunction before the actual death of the cells. Whether these dying neurons can be rescued and return to a normal, functional state is uncertain. In the present study, we explored the reversibility of the neuronal cell death pathway at various stages by monitoring the dynamics of single cells with high-resolution live-cell spinning disk confocal microscopy in an in vitro neuronal cell death model. We exposed differentiated neuronal PC12 cells to ethanol as our cell death model. Results showed that exposure to 5% ethanol for 24 h induced cell death in >70% of the cells. Ethanol treatment for 3 h already induced cellular changes and damage such as reactive oxygen species generation, elevation of intracellular Ca2+ level, phosphatidylserine exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation and membrane potential loss, and retraction of neurites. These phenomena are often associated with programmed cell death. Importantly, after removing ethanol and further culturing these damaged cells in fresh culture medium, cells recovered from all these cell injuries and generated new neurites. Moreover, results indicated that this recovery was not dependent on exogenous NGF and other growth factors in the cell culture medium. Overall, our results suggest that targeting dying neurons can be an effective therapeutic strategy in neurodegenerative diseases.

Structure-Based Cyclic Glycoprotein Ibα-Derived Peptides Interfering with von Willebrand Factor-Binding, Affecting Platelet Aggregation under Shear.

The plasmatic von Willebrand factor (VWF) circulates in a compact form unable to bind platelets. Upon shear stress, the VWF A1 domain is exposed, allowing VWF-binding to platelet glycoprotein Ib-V-IX (GPIbα chain). For a better understanding of the role of this interaction in cardiovascular disease, molecules are needed to specifically interfere with the opened VWF A1 domain interaction with GPIbα. Therefore, we in silico designed and chemically synthetized stable cyclic peptides interfering with the platelet-binding of the VWF A1 domain per se or complexed with botrocetin. Selected peptides (26-34 amino acids) with the lowest-binding free energy were: the monocyclic mono- vOn Willebrand factoR-GPIbα InTerference (ORbIT) peptide and bicyclic bi-ORbIT peptide. Interference of the peptides in the binding of VWF to GPIb-V-IX interaction was retained by flow cytometry in comparison with the blocking of anti-VWF A1 domain antibody CLB-RAg35. In collagen and VWF-dependent whole-blood thrombus formation at a high shear rate, CLB-RAg35 suppressed stable platelet adhesion as well as the formation of multilayered thrombi. Both peptides phenotypically mimicked these changes, although they were less potent than CLB-RAg35. The second-round generation of an improved peptide, namely opt-mono-ORbIT (28 amino acids), showed an increased inhibitory activity under flow. Accordingly, our structure-based design of peptides resulted in physiologically effective peptide-based inhibitors, even for convoluted complexes such as GPIbα-VWF A1.

A collagen-binding protein enables molecular imaging of kidney fibrosis in vivo.

Pathological deposition of collagen is a hallmark of kidney fibrosis. To illustrate this process we employed multimodal optical imaging to visualize and quantify collagen deposition in murine models of kidney fibrosis (ischemia-reperfusion or unilateral ureteral obstruction) using the collagen-binding adhesion protein CNA35. For in vivo imaging, we used hybrid computed tomography-fluorescence molecular tomography and CNA35 labeled with the near-infrared fluorophore Cy7. Upon intravenous injection, CNA35-Cy7 accumulation was significantly higher in fibrotic compared to non-fibrotic kidneys. This difference was not detected for a non-specific scrambled version of CNA35-Cy7. Ex vivo, on kidney sections of mice and patients with renal fibrosis, CNA35-FITC co-localized with fibrotic collagen type I and III, but not with the basement membrane collagen type IV. Following intravenous injection, CNA35-FITC bound to both interstitial and perivascular fibrotic areas. In line with this perivascular accumulation, we observed significant perivascular fibrosis in the mouse models and in biopsy sections from patients with chronic kidney disease using computer-based morphometry quantification. Thus, molecular imaging of collagen using CNA35 enabled specific non-invasive quantification of kidney fibrosis. Collagen imaging revealed significant perivascular fibrosis as a consistent component next to the more commonly assessed interstitial fibrosis. Our results lay the basis for further probe and protocol optimization towards the clinical translation of molecular imaging of kidney fibrosis.

The natural course of pregnancies in women with primary atypical haemolytic uraemic syndrome and asymptomatic relatives.

Pregnancy has been linked to various microangiopathies, including primary atypical haemolytic uraemic syndrome (aHUS). Complement dysregulation, often linked to rare variants in complement genes, is key for primary aHUS to manifest and may play a role in pregnancy complications of the mother and fetus. The burden of such complications is unknown, making counselling of women with primary aHUS and asymptomatic relatives difficult. We analyzed the maternal and fetal outcomes of 39 pregnancies from 17 women with primary aHUS and two asymptomatic relatives. Seven out of 39 pregnancies were complicated by pregnancy-associated aHUS. Five out of 32 pregnancies not linked to pregnancy-associated aHUS were complicated by pre-eclampsia or HELLP. Rare genetic variants were identified in 10 women (asymptomatic relatives, n = 2) who had a total of 14 pregnancies, including 10 uncomplicated pregnancies. Thirty-five out of 39 pregnancies resulted in live birth. Eight out of 19 women had progressed to end-stage kidney disease, with an incidence of 2·95 (95% confidence interval, 1·37-5·61) per 100 person-years after the first pregnancy. Thus, we emphasized the frequency of successful pregnancies in women with primary aHUS and asymptomatic relatives. Pregnancies should be monitored closely. Rare genetic variants cannot predict the risk of a given pregnancy.

Extracellular Vesicles in CNS Developmental Disorders.

The central nervous system (CNS) is the most complex structure in the body, consisting of multiple cell types with distinct morphology and function. Development of the neuronal circuit and its function rely on a continuous crosstalk between neurons and non-neural cells. It has been widely accepted that extracellular vesicles (EVs), mainly exosomes, are effective entities responsible for intercellular CNS communication. They contain membrane and cytoplasmic proteins, lipids, non-coding RNAs, microRNAs and mRNAs. Their cargo modulates gene and protein expression in recipient cells. Several lines of evidence indicate that EVs play a role in modifying signal transduction with subsequent physiological changes in neurogenesis, gliogenesis, synaptogenesis and network circuit formation and activity, as well as synaptic pruning and myelination. Several studies demonstrate that neural and non-neural EVs play an important role in physiological and pathological neurodevelopment. The present review discusses the role of EVs in various neurodevelopmental disorders and the prospects of using EVs as disease biomarkers and therapeutics.

Proteomic analysis of neutrophils in ANCA-associated vasculitis reveals a dysregulation in proteinase 3-associated proteins such as annexin-A1 involved in apoptotic cell clearance.

Granulomatosis with polyangiitis (GPA) is an autoimmune vasculitis associated with anti-neutrophil-cytoplasmic antibodies (ANCA) against proteinase 3 leading to kidney damage. Neutrophils from those patients have increased expression of membrane proteinase 3 during apoptosis. Here we examined whether neutrophils from patients with GPA have dysregulated protein expressions associated with apoptosis. A global proteomic analysis was performed comparing neutrophils from patients with GPA, with healthy individuals under basal conditions and during apoptosis. At disease onset, the cytosolic proteome of neutrophils of patients with GPA before treatment was significantly different from healthy controls, and this dysregulation was more pronounced following ex vivo apoptosis. Proteins involved in cell death/survival were altered in neutrophils of patients with GPA. Several proteins identified were PR3-binding partners involved in the clearance of apoptotic cells, namely calreticulin, annexin-A1 and phospholipid scramblase 1. These proteins form a platform at the membrane of apoptotic neutrophils in patients with GPA but not healthy individuals and this was associated with the clinical presentation of GPA. Thus, our study shows that neutrophils from patients with GPA have an intrinsic dysregulation in proteins involved in apoptotic cell clearance, which could contribute to the unabated inflammation and autoimmunity in GPA. Hence, harnessing these dysregulated pathways could lead to novel biomarkers and targeted therapeutic opportunities to treat kidney disease.

C5b9 Formation on Endothelial Cells Reflects Complement Defects among Patients with Renal Thrombotic Microangiopathy and Severe Hypertension.

Background Severe hypertension can induce thrombotic microangiopathy (TMA) in the renal vasculature, the occurrence of which has been linked to mechanical stress to the endothelium. Complement defects may be the culprit of disease in patients who present with severe renal disease and often progress to ESRD, despite BP control.Methods We studied a well defined cohort of 17 patients with hypertension-associated TMA to define the prevalence of complement defects by a specific ex vivo serum-based microvascular endothelial cell assay.Results Compared with normal human serum and samples from patients with hypertensive arterionephrosclerosis, 14 of 16 (87.5%) serum samples collected at presentation from 16 patients with hypertension-associated TMA induced abnormal C5b9 formation on microvascular endothelial cells. We detected rare variants in complement genes in eight of 17 (47%) patients. ESRD occurred in 14 of 17 (82%) patients, and recurrent TMA after transplant occurred in seven of 11 (64%) donor kidneys. Eculizumab improved the renal function in three patients and prevented TMA recurrence in an allograft recipient.Conclusions These observations point to complement defects as the key causative factor of ESRD and recurrent TMA after transplant in patients presenting with severe hypertension. Complement defects can be identified by measurements of complement activation on microvascular endothelial cells, which should substantially influence treatment and prognosis.

Patients with hypertension-associated thrombotic microangiopathy may present with complement abnormalities.

Thrombotic microangiopathy (TMA) is a pattern of endothelial damage that can be found in association with diverse clinical conditions such as malignant hypertension. Although the pathophysiological mechanisms differ, accumulating evidence links complement dysregulation to various TMA syndromes and in particular the atypical hemolytic uremic syndrome. Here, we evaluated the role of complement in nine consecutive patients with biopsy-proven renal TMA attributed to severe hypertension. Profound hematologic symptoms of TMA were uncommon. In six out of nine patients, we found mutations C3 in three, CFI in one, CD46 in one, and/or CFH in two patients either with or without the risk CFH-H3 haplotype in four patients. Elevated levels of the soluble C5b-9 and renal deposits of C3c and C5b-9 along the vasculature and/or glomerular capillary wall, confirmed complement activation in vivo. In contrast to patients without genetic defects, patients with complement defects invariably progressed to end-stage renal disease, and disease recurrence after kidney transplantation seems common. Thus, a subset of patients with hypertension-associated TMA falls within the spectrum of complement-mediated TMA, the prognosis of which is poor. Hence, testing for genetic complement abnormalities is warranted in patients with severe hypertension and TMA on renal biopsy to adopt suitable treatment options and prophylactic measures.

Vascular smooth muscle cell calcification is mediated by regulated exosome secretion.

RATIONALE: Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. OBJECTIVE: The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. METHODS AND RESULTS: Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. CONCLUSIONS: This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.

Nonanticoagulant heparin prevents histone-mediated cytotoxicity in vitro and improves survival in sepsis.

Extracellular histones are considered to be major mediators of death in sepsis. Although sepsis is a condition that may benefit from low-dose heparin administration, medical doctors need to take into consideration the potential bleeding risk in sepsis patients who are already at increased risk of bleeding due to a consumption coagulopathy. Here, we show that mechanisms that are independent of the anticoagulant properties of heparin may contribute to the observed beneficial effects of heparin in the treatment of sepsis patients. We show that nonanticoagulant heparin, purified from clinical grade heparin, binds histones and prevents histone-mediated cytotoxicity in vitro and reduces mortality from sterile inflammation and sepsis in mouse models without increasing the risk of bleeding. Our results demonstrate that administration of nonanticoagulant heparin is a novel and promising approach that may be further developed to treat patients suffering from sepsis.

AnxA5 reduces plaque inflammation of advanced atherosclerotic lesions in apoE(-/-) mice.

Annexin A5 (AnxA5) exerts anti-inflammatory, anticoagulant and anti-apoptotic effects through binding cell surface expressed phosphatidylserine. The actions of AnxA5 on atherosclerosis are incompletely understood. We investigated effects of exogenous AnxA5 on plaque morphology and phenotype of advanced atherosclerotic lesions in apoE(-/-) mice. Advanced atherosclerotic lesions were induced in 12 weeks old Western type diet fed apoE(-/-) mice using a collar placement around the carotid artery. After 5 weeks mice were injected either with AnxA5 (n = 8) or vehicle for another 4 weeks. AnxA5 reduced plaque macrophage content both in the intima (59% reduction, P < 0.05) and media (73% reduction, P < 0.01) of advanced atherosclerotic lesions of the carotid artery. These findings corroborated with advanced lesions of the aortic arch, where a 67% reduction in plaque macrophage content was observed with AnxA5 compared to controls (P < 0.01). AnxA5 did not change lesion extension, plaque apoptosis, collagen content, smooth muscle cell content or acellular plaque composition after 4 weeks of treatment as determined by immunohistochemistry in advanced carotid lesions. In vitro, AnxA5 exhibited anti-inflammatory effects in macrophages and a flow chamber based assay demonstrated that AnxA5 significantly inhibited capture, rolling, adhesion as well as transmigration of peripheral blood mononuclear cells on a TNF-α-activated endothelial cell layer. In conclusion, short-term treatment with AnxA5 reduces plaque inflammation of advanced lesions in apoE(-/-) mice likely through interfering with recruitment and activation of monocytes to the inflamed lesion site. Suppressing chronic inflammation by targeting exposed phosphatidylserine may become a viable strategy to treat patients suffering from advanced atherosclerosis.