High phosphate and calcium induce osteoblastic phenotype switching and calcification of corneal epithelial cells in a Runx2-dependent and synergistic manner; a possible mechanism of chronic kidney disease-associated corneal calcification.

Patients with advanced chronic kidney disease (CKD) have elevated circulating calcium × phosphate product levels and exhibit soft tissue calcification. Besides the cardiovascular system, calcification is commonly observed in the cornea in CKD patients on hemodialysis. Cardiovascular calcification is a cell-mediated, highly regulated process, and we hypothesized that a similar regulatory mechanism is implicated in corneal calcification with the involvement of corneal epithelial cells (CECs). We established a mouse model of CKD-associated corneal calcification by inducing CKD in DBA/2J mice with an adenine and high phosphate diet. CKD was associated with aorta and corneal calcification as detected by OsteoSense staining and corneal Ca measurement (1.67-fold elevation, p < 0.001). In vitro, excess phosphate and Ca induced human CEC calcification in a dose-dependent and synergistic manner, without any influence on cell viability. High phosphate and Ca-containing osteogenic medium (OM; 2.5 mmol/L excess phosphate and 0.6 mmol/L excess Ca over control) increased the protein expression of Runx2 and induced its nuclear translocation. OM increased the expression of the bone-specific Ca-binding protein osteocalcin (130-fold increase, p < 0.001). Silencing of Runx2 attenuated OM-induced CEC calcification. Immunohistology revealed upregulation of Runx2 and overlapping between the Runx2 and the Alizarin red positive areas of calcification in the cornea of CKD mice. This work sheds light on the mechanism of CKD-induced corneal calcification and provides tools to test calcification inhibitors for the prevention of this detrimental process.

Targeting circulating labile heme as a defense strategy against malaria.

Severe presentations of malaria emerge as Plasmodium (P.) spp. parasites invade and lyse red blood cells (RBC), producing extracellular hemoglobin (HB), from which labile heme is released. Here, we tested whether scavenging of extracellular HB and/or labile heme, by haptoglobin (HP) and/or hemopexin (HPX), respectively, counter the pathogenesis of severe presentations of malaria. We found that circulating labile heme is an independent risk factor for cerebral and non-cerebral presentations of severe P. falciparum malaria in children. Labile heme was negatively correlated with circulating HP and HPX, which were, however, not risk factors for severe P. falciparum malaria. Genetic Hp and/or Hpx deletion in mice led to labile heme accumulation in plasma and kidneys, upon Plasmodium infection This was associated with higher incidence of mortality and acute kidney injury (AKI) in ageing but not adult Plasmodium-infected mice, and was corroborated by an inverse correlation between heme and HPX with serological markers of AKI in P. falciparum malaria. In conclusion, HP and HPX act in an age-dependent manner to prevent the pathogenesis of severe presentation of malaria in mice and presumably in humans.

High glucose promotes osteogenic differentiation of human lens epithelial cells through hypoxia-inducible factor (HIF) activation.

Cataract, a leading cause of blindness, is characterised by lens opacification. Type 2 diabetes is associated with a two- to fivefold higher prevalence of cataracts. The risk of cataract formation increases with the duration of diabetes and the severity of hyperglycaemia. Hydroxyapatite deposition is present in cataractous lenses that could be the consequence of osteogenic differentiation and calcification of lens epithelial cells (LECs). We hypothesised that hyperglycaemia might promote the osteogenic differentiation of human LECs (HuLECs). Osteogenic medium (OM) containing excess phosphate and calcium with normal (1 g/L) or high (4.5 g/L) glucose was used to induce HuLEC calcification. High glucose accelerated and intensified OM-induced calcification of HuLECs, which was accompanied by hyperglycaemia-induced upregulation of the osteogenic markers Runx2, Sox9, alkaline phosphatase and osteocalcin, as well as nuclear translocation of Runx2. High glucose-induced calcification was abolished in Runx2-deficient HuLECs. Additionally, high glucose stabilised the regulatory alpha subunits of hypoxia-inducible factor 1 (HIF-1), triggered nuclear translocation of HIF-1α and increased the expression of HIF-1 target genes. Gene silencing of HIF-1α or HIF-2α attenuated hyperglycaemia-induced calcification of HuLECs, while hypoxia mimetics (desferrioxamine, CoCl2) enhanced calcification of HuLECs under normal glucose conditions. Overall, this study suggests that high glucose promotes HuLEC calcification via Runx2 and the activation of the HIF-1 signalling pathway. These findings may provide new insights into the pathogenesis of diabetic cataracts, shedding light on potential factors for intervention to treat this sight-threatening condition.

Hypoxia-inducible factor activation promotes osteogenic transition of valve interstitial cells and accelerates aortic valve calcification in a mice model of chronic kidney disease.

Introduction: Valve calcification (VC) is a widespread complication in chronic kidney disease (CKD) patients. VC is an active process with the involvement of in situ osteogenic transition of valve interstitial cells (VICs). VC is accompanied by the activation of hypoxia inducible factor (HIF) pathway, but the role of HIF activation in the calcification process remains undiscovered. Methods and result: Using in vitro and in vivo approaches we addressed the role of HIF activation in osteogenic transition of VICs and CKD-associated VC. Elevation of osteogenic (Runx2, Sox9) and HIF activation markers (HIF-1α and HIF-2α) and VC occurred in adenine-induced CKD mice. High phosphate (Pi) induced upregulation of osteogenic (Runx2, alkaline-phosphatase, Sox9, osteocalcin) and hypoxia markers (HIF-1α, HIF-2α, Glut-1), and calcification in VICs. Down-regulation of HIF-1α and HIF-2α inhibited, whereas further activation of HIF pathway by hypoxic exposure (1% O2) or hypoxia mimetics [desferrioxamine, CoCl2, Daprodustat (DPD)] promoted Pi-induced calcification of VICs. Pi augmented the formation of reactive oxygen species (ROS) and decreased viability of VICs, whose effects were further exacerbated by hypoxia. N-acetyl cysteine inhibited Pi-induced ROS production, cell death and calcification under both normoxic and hypoxic conditions. DPD treatment corrected anemia but promoted aortic VC in the CKD mice model. Discussion: HIF activation plays a fundamental role in Pi-induced osteogenic transition of VICs and CKD-induced VC. The cellular mechanism involves stabilization of HIF-1α and HIF-2α, increased ROS production and cell death. Targeting the HIF pathways may thus be investigated as a therapeutic approach to attenuate aortic VC.

The transcriptional control of the VEGFA-VEGFR1 (FLT1) axis in alternatively polarized murine and human macrophages.

Introduction: Macrophages significantly contribute to the regulation of vessel formation under physiological and pathological conditions. Although the angiogenesis-regulating role of alternatively polarized macrophages is quite controversial, a growing number of evidence shows that they can participate in the later phases of angiogenesis, including vessel sprouting and remodeling or regression. However, the epigenetic and transcriptional regulatory mechanisms controlling this angiogenesis-modulating program are not fully understood. Results: Here we show that IL-4 can coordinately regulate the VEGFA-VEGFR1 (FLT1) axis via simultaneously inhibiting the proangiogenic Vegfa and inducing the antiangiogenic Flt1 expression in murine bone marrow-derived macrophages, which leads to the attenuated proangiogenic activity of alternatively polarized macrophages. The IL-4-activated STAT6 and IL-4-STAT6 signaling pathway-induced EGR2 transcription factors play a direct role in the transcriptional regulation of the Vegfa-Flt1 axis. We demonstrated that this phenomenon is not restricted to the murine bone marrow-derived macrophages, but can also be observed in different murine tissue-resident macrophages ex vivo and parasites-elicited macrophages in vivo with minor cell type-specific differences. Furthermore, IL-4 exposure can modulate the hypoxic response of genes in both murine and human macrophages leading to a blunted Vegfa/VEGFA and synergistically induced Flt1/FLT1 expression. Discussion: Our findings establish that the IL-4-activated epigenetic and transcriptional program can determine angiogenesis-regulating properties in alternatively polarized macrophages under normoxic and hypoxic conditions.

The mechanosensitive Piezo1 channels contribute to the arterial medial calcification.

Vascular calcification (VC) is associated with a number of cardiovascular diseases, as well as chronic kidney disease. The role of smooth muscle cells (SMC) has already been widely explored in VC, as has the role of intracellular Ca2+ in regulating SMC function. Increased intracellular calcium concentration ([Ca2+]i) in vascular SMC has been proposed to stimulate VC. However, the contribution of the non-selective Piezo1 mechanosensitive cation channels to the elevation of [Ca2+]i, and consequently to the process of VC has never been examined. In this work the essential contribution of Piezo1 channels to arterial medial calcification is demonstrated. The presence of Piezo1 was proved on human aortic smooth muscle samples using immunohistochemistry. Quantitative PCR and Western blot analysis confirmed the expression of the channel on the human aortic smooth muscle cell line (HAoSMC). Functional measurements were done on HAoSMC under control and calcifying condition. Calcification was induced by supplementing the growth medium with inorganic phosphate (1.5 mmol/L, pH 7.4) and calcium (CaCl2, 0.6 mmol/L) for 7 days. Measurement of [Ca2+]i using fluorescent Fura-2 dye upon stimulation of Piezo1 channels (either by hypoosmolarity, or Yoda1) demonstrated significantly higher calcium transients in calcified as compared to control HAoSMCs. The expression of mechanosensitive Piezo1 channel is augmented in calcified arterial SMCs leading to a higher calcium influx upon stimulation. Activation of the channel by Yoda1 (10 µmol/L) enhanced calcification of HAoSMCs, while Dooku1, which antagonizes the effect of Yoda1, reduced this amplification. Application of Dooku1 alone inhibited the calcification. Knockdown of Piezo1 by siRNA suppressed the calcification evoked by Yoda1 under calcifying conditions. Our results demonstrate the pivotal role of Piezo1 channels in arterial medial calcification.

The Effect of Activated FXIII, a Transglutaminase, on Vascular Smooth Muscle Cells.

Plasma factor XIII (pFXIII) is a heterotetramer of FXIII-A and FXIII-B subunits. The cellular form (cFXIII), a dimer of FXIII-A, is present in a number of cell types. Activated FXIII (FXIIIa), a transglutaminase, plays an important role in clot stabilization, wound healing, angiogenesis and maintenance of pregnancy. It has a direct effect on vascular endothelial cells and fibroblasts, which have been implicated in the development of atherosclerotic plaques. Our aim was to explore the effect of FXIIIa on human aortic smooth muscle cells (HAoSMCs), another major cell type in the atherosclerotic plaque. Osteoblastic transformation induced by Pi and Ca2+ failed to elicit the expression of cFXIII in HAoSMCs. EZ4U, CCK-8 and CytoSelect Wound Healing assays were used to investigate cell proliferation and migration. The Sircol Collagen Assay Kit was used to monitor collagen secretion. Thrombospondin-1 (TSP-1) levels were measured by ELISA. Cell-associated TSP-1 was detected by the immunofluorescence technique. The TSP-1 mRNA level was estimated by RT-qPCR. Activated recombinant cFXIII (rFXIIIa) increased cell proliferation and collagen secretion. In parallel, a 67% decrease in TSP-1 concentration in the medium and a 2.5-fold increase in cells were observed. TSP-1 mRNA did not change significantly. These effects of FXIIIa might contribute to the pathogenesis of atherosclerotic plaques.

Activation of Nrf2/HO-1 Antioxidant Pathway by Heme Attenuates Calcification of Human Lens Epithelial Cells.

Cataract, an opacification in the crystalline lens, is a leading cause of blindness. Deposition of hydroxyapatite occurs in a cataractous lens that could be the consequence of osteogenic differentiation of lens epithelial cells (LECs). Nuclear factor erythroid 2-related factor 2 (Nrf2) controls the transcription of a wide range of cytoprotective genes. Nrf2 upregulation attenuates cataract formation. Here we aimed to investigate the effect of Nrf2 system upregulation in LECs calcification. We induced osteogenic differentiation of human LECs (HuLECs) with increased phosphate and calcium-containing osteogenic medium (OM). OM-induced calcium and osteocalcin deposition in HuLECs. We used heme to activate Nrf2, which strongly upregulated the expression of Nrf2 and heme oxygenase-1 (HO-1). Heme-mediated Nrf2 activation was dependent on the production of reactive oxygens species. Heme inhibited Ca deposition, and the OM-induced increase of osteogenic markers, RUNX2, alkaline phosphatase, and OCN. Anti-calcification effect of heme was lost when the transcriptional activity of Nrf2 or the enzyme activity of HO-1 was blocked with pharmacological inhibitors. Among products of HO-1 catalyzed heme degradation iron mimicked the anti-calcification effect of heme. We concluded that heme-induced upregulation of the Nrf2/HO-1 system inhibits HuLECs calcification through the liberation of heme iron.

Daprodustat Accelerates High Phosphate-Induced Calcification Through the Activation of HIF-1 Signaling.

Aims: Chronic kidney disease (CKD) is frequently associated with other chronic diseases including anemia. Daprodustat (DPD) is a prolyl hydroxylase inhibitor, a member of a family of those new generation drugs that increase erythropoiesis via activation of the hypoxia-inducible factor 1 (HIF-1) pathway. Previous studies showed that HIF-1 activation is ultimately linked to acceleration of vascular calcification. We aimed to investigate the effect of DPD on high phosphate-induced calcification. Methods and Results: We investigated the effect of DPD on calcification in primary human aortic vascular smooth muscle cells (VSMCs), in mouse aorta rings, and an adenine and high phosphate-induced CKD murine model. DPD stabilized HIF-1α and HIF-2α and activated the HIF-1 pathway in VSMCs. Treatment with DPD increased phosphate-induced calcification in cultured VSMCs and murine aorta rings. Oral administration of DPD to adenine and high phosphate-induced CKD mice corrected anemia but increased aortic calcification as assessed by osteosense staining. The inhibition of the transcriptional activity of HIF-1 by chetomin or silencing of HIF-1α attenuated the effect of DPD on VSMC calcification. Conclusion: Clinical studies with a long follow-up period are needed to evaluate the possible risk of sustained activation of HIF-1 by DPD in accelerating medial calcification in CKD patients with hyperphosphatemia.

Preterm Intraventricular Hemorrhage-Induced Inflammatory Response in Human Choroid Plexus Epithelial Cells.

Following an intraventricular hemorrhage (IVH), red blood cell lysis and hemoglobin (Hb) oxidation with the release of heme can cause sterile neuroinflammation. In this study, we measured Hb derivates and cellular adhesion molecules ICAM-1 and VCAM-1 with cell-free miRNAs in cerebrospinal fluid (CSF) samples obtained from Grade-III and Grade-IV preterm IVH infants (IVH-III and IVH-IV, respectively) at multiple time points between days 0-60 after the onset of IVH. Furthermore, human choroid plexus epithelial cells (HCPEpiCs) were incubated with IVH and non-IVH CSF (10 v/v %) for 24 h in vitro to investigate the IVH-induced inflammatory response that was investigated via: (i) HMOX1, IL8, VCAM1, and ICAM1 mRNAs as well as miR-155, miR-223, and miR-181b levels by RT-qPCR; (ii) nuclear translocation of the NF-κB p65 subunit by fluorescence microscopy; and (iii) reactive oxygen species (ROS) measurement. We found a time-dependent alteration of heme, IL-8, and adhesion molecules which revealed a prolonged elevation in IVH-IV vs. IVH-III with higher miR-155 and miR-181b expression at days 41-60. Exposure of HCPEpiCs to IVH CSF samples induced HMOX1, IL8, and ICAM1 mRNA levels along with increased ROS production via the NF-κB pathway activation but without cell death, as confirmed by the cell viability assay. Additionally, the enhanced intracellular miR-155 level was accompanied by lower miR-223 and miR-181b expression in HCPEpiCs after CSF treatment. Overall, choroid plexus epithelial cells exhibit an abnormal cell phenotype after interaction with pro-inflammatory CSF of IVH origin which may contribute to the development of later clinical complications in preterm IVH.

Heme-Mediated Activation of the Nrf2/HO-1 Axis Attenuates Calcification of Valve Interstitial Cells.

Calcific aortic valve stenosis (CAVS) is a heart disease characterized by the progressive fibro-calcific remodeling of the aortic valves, an actively regulated process with the involvement of the reactive oxygen species-mediated differentiation of valvular interstitial cells (VICs) into osteoblast-like cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the expression of a variety of antioxidant genes, and plays a protective role in valve calcification. Heme oxygenase-1 (HO-1), an Nrf2-target gene, is upregulated in human calcified aortic valves. Therefore, we investigated the effect of Nrf2/HO-1 axis in VIC calcification. We induced osteogenic differentiation of human VICs with elevated phosphate and calcium-containing osteogenic medium (OM) in the presence of heme. Heme inhibited Ca deposition and OM-induced increase in alkaline phosphatase and osteocalcin (OCN) expression. Heme induced Nrf2 and HO-1 expression in VICs. Heme lost its anti-calcification potential when we blocked transcriptional activity Nrf2 or enzyme activity of HO-1. The heme catabolism products bilirubin, carbon monoxide, and iron, and also ferritin inhibited OM-induced Ca deposition and OCN expression in VICs. This study suggests that heme-mediated activation of the Nrf2/HO-1 pathway inhibits the calcification of VICs. The anti-calcification effect of heme is attributed to the end products of HO-1-catalyzed heme degradation and ferritin.

Regulation of Vascular Calcification by Reactive Oxygen Species.

Vascular calcification is the deposition of hydroxyapatite crystals in the medial or intimal layers of arteries that is usually associated with other pathological conditions including but not limited to chronic kidney disease, atherosclerosis and diabetes. Calcification is an active, cell-regulated process involving the phenotype transition of vascular smooth muscle cells (VSMCs) from contractile to osteoblast/chondrocyte-like cells. Diverse triggers and signal transduction pathways have been identified behind vascular calcification. In this review, we focus on the role of reactive oxygen species (ROS) in the osteochondrogenic phenotype switch of VSMCs and subsequent calcification. Vascular calcification is associated with elevated ROS production. Excessive ROS contribute to the activation of certain osteochondrogenic signal transduction pathways, thereby accelerating osteochondrogenic transdifferentiation of VSMCs. Inhibition of ROS production and ROS scavengers and activation of endogenous protective mechanisms are promising therapeutic approaches in the prevention of osteochondrogenic transdifferentiation of VSMCs and subsequent vascular calcification. The present review discusses the formation and actions of excess ROS in different experimental models of calcification, and the potential of ROS-lowering strategies in the prevention of this deleterious condition.

Elevated Pro-Inflammatory Cell-Free MicroRNA Levels in Cerebrospinal Fluid of Premature Infants after Intraventricular Hemorrhage.

Intraventricular hemorrhage (IVH) represents a high risk of neonatal mortality and later neurodevelopmental impairment in prematurity. IVH is accompanied with inflammation, hemolysis, and extracellular hemoglobin (Hb) oxidation. However, microRNA (miRNA) expression in cerebrospinal fluid (CSF) of preterm infants with IVH has been unknown. Therefore, in the present study, candidate pro-inflammatory cell-free miRNAs were analyzed in CSF samples from 47 preterm infants with grade III or IV IVH vs. clinical controls (n = 14). miRNAs were quantified by RT-qPCR, normalized to "spike-in" cel-miR-39. Oxidized Hb and total heme levels were determined by spectrophotometry as well as IL-8, VCAM-1, ICAM-1, and E-selectin concentrations by ELISA. To reveal the origin of the investigated miRNAs, controlled hemolysis experiments were performed in vitro; in addition, human choroid plexus epithelial cell (HCPEpiC) cultures were treated with metHb, ferrylHb, heme, or TNF-α to replicate IVH-triggered cellular conditions. Levels of miR-223, miR-155, miR-181b, and miR-126 as well as Hb metabolites along with IL-8 were elevated in CSF after the onset of IVH vs. controls. Significant correlations were observed among the miRNAs, oxidized Hb forms, and the soluble adhesion molecules. During the post-IVH follow-up, attenuated expression of miRNAs and protein biomarkers in CSF was observed upon elimination of Hb metabolites. These miRNAs remained unaffected by a series of artificially induced hemolysis, which excluded red blood cells as their origin, while stimulation of HCPEpiCs with oxidized Hb fractions and heme resulted in increased extracellular miRNA levels in the cell culture supernatant. Overall, the hemorrhage-induced CSF miRNAs reflected inflammatory conditions as potential biomarkers in preterm IVH.

Pro-inflammatory Actions of Heme and Other Hemoglobin-Derived DAMPs.

Damage associated molecular patterns (DAMPs) are endogenous molecules originate from damaged cells and tissues with the ability to trigger and/or modify innate immune responses. Upon hemolysis hemoglobin (Hb) is released from red blood cells (RBCs) to the circulation and give a rise to the production of different Hb redox states and heme which can act as DAMPs. Heme is the best characterized Hb-derived DAMP that targets different immune and non-immune cells. Heme is a chemoattractant, activates the complement system, modulates host defense mechanisms through the activation of innate immune receptors and the heme oxygenase-1/ferritin system, and induces innate immune memory. The contribution of oxidized Hb forms is much less studied, but some evidence show that these species might play distinct roles in intravascular hemolysis-associated pathologies independently of heme release. This review aims to summarize our current knowledge about the formation and pro-inflammatory actions of heme and other Hb-derived DAMPs.

Formation and Detection of Highly Oxidized Hemoglobin Forms in Biological Fluids during Hemolytic Conditions.

Hemolytic diseases are characterized by an accelerated breakdown of red blood cells (RBCs) and the release of hemoglobin (Hb). Following, RBC lysis Hb oxidation occurs with the formation of different redox states of Hb (metHb and ferrylHb) and the release of heme. ferrylHb is unstable and decomposes to metHb with the concomitant formation of globin radicals and eventually covalently crosslinked Hb multimers. The goal of the present study was to determine the concentrations of the different redox states of Hb in biological samples during hemolytic conditions. We used plasma and urine samples of mice with intravascular hemolysis and human cerebrospinal fluid (CSF) samples following intraventricular hemorrhage. Because ferrylHb is highly unstable, we also addressed the fate of this species. metHb and free heme time-dependently accumulate in plasma and CSF samples following intravascular hemolysis and intraventricular hemorrhage, respectively. ferrylHb is hardly detectable in the biological samples during hemolytic conditions. Under in vitro conditions, ferrylHb decomposes quickly to metHb, which process is associated with the formation of covalently crosslinked Hb multimers. We detected these covalently crosslinked Hb multimers in plasma, urine, and CSF samples during hemolytic conditions. Because globin modification is specific for these Hb forms, we propose to call this heterogeneous form of Hb produced during ferrylHb decomposition as globin-modified oxidized Hb (gmoxHb). Understanding the formation and the contribution of gmoxHb species to the pathogenesis of hemolytic conditions could have therapeutic implications in the treatment of hemolytic diseases.

The Role of Hemoglobin Oxidation Products in Triggering Inflammatory Response Upon Intraventricular Hemorrhage in Premature Infants.

Intraventricular hemorrhage (IVH) is a frequent complication of prematurity that is associated with high neonatal mortality and morbidity. IVH is accompanied by red blood cell (RBC) lysis, hemoglobin (Hb) oxidation, and sterile inflammation. Here we investigated whether extracellular Hb, metHb, ferrylHb, and heme contribute to the inflammatory response after IVH. We collected cerebrospinal fluid (CSF) (n = 20) from premature infants with grade III IVH at different time points after the onset of IVH. Levels of Hb, metHb, total heme, and free heme were the highest in CSF samples obtained between days 0 and 20 after the onset of IVH and were mostly non-detectable in CSF collected between days 41 and 60 of post-IVH. Besides Hb monomers, we detected cross-linked Hb dimers and tetramers in post-IVH CSF samples obtained in days 0-20 and 21-40, but only Hb tetramers were present in CSF samples obtained after 41-60 days. Vascular cell adhesion molecule-1 (VCAM-1) and interleukin-8 (IL-8) levels were higher in CSF samples obtained between days 0 and 20 than in CSF collected between days 41 and 60 of post-IVH. Concentrations of VCAM-1, intercellular adhesion molecule-1 (ICAM-1), and IL-8 strongly correlated with total heme levels in CSF. Applying the identified heme sources on human brain microvascular endothelial cells revealed that Hb oxidation products and free heme contribute to the inflammatory response. We concluded that RBC lysis, Hb oxidation, and heme release are important components of the inflammatory response in IVH. Pharmacological interventions targeting cell-free Hb, Hb oxidation products, and free heme could have potential to limit the neuroinflammatory response following IVH.

Pharmacokinetic Properties of Fluorescently Labelled Hydroxypropyl-Beta-Cyclodextrin.

2-Hydroxypropyl-beta-cyclodextrin (HPBCD) is utilized in the formulation of pharmaceutical products and recently orphan designation was granted for the treatment of Niemann-Pick disease, type C. The exact mechanism of HPBCD action and side effects are not completely explained. We used fluorescently labelled hydroxypropyl-beta-cyclodextrin (FITC-HPBCD) to study its pharmacokinetic parameters in mice and compare with native HPBCD data. We found that FITC-HPBCD has fast distribution and elimination, similar to HPBCD. Interestingly animals could be divided into two groups, where the pharmacokinetic parameters followed or did not follow the two-compartment, first-order kinetic model. Tissue distribution studies revealed, that a significant amount of FITC-HPBCD could be detected in kidneys after 60 min treatment, due to its renal excretion. Ex vivo fluorescent imaging showed that fluorescence could be measured in lung, liver, brain and spleen after 30 min of treatment. To model the interaction and cellular distribution of FITC-HPBCD in the wall of blood vessels, we treated human umbilical vein endothelial cells (HUVECs) with FITC-HPBCD and demonstrated for the first time that this compound could be detected in the cytoplasm in small vesicles after 30 min of treatment. FITC-HPBCD has similar pharmacokinetic to HPBCD and can provide new information to the detailed mechanism of action of HPBCD.

Hypoxia Triggers Osteochondrogenic Differentiation of Vascular Smooth Muscle Cells in an HIF-1 (Hypoxia-Inducible Factor 1)-Dependent and Reactive Oxygen Species-Dependent Manner.

Objective- Vascular calcification is associated with high risk of cardiovascular events and mortality. Osteochondrogenic differentiation of vascular smooth muscle cells (VSMCs) is the major cellular mechanism underlying vascular calcification. Because tissue hypoxia is a common denominator in vascular calcification, we investigated whether hypoxia per se triggers osteochondrogenic differentiation of VSMCs. Approach and Results- We studied osteochondrogenic differentiation of human aorta VSMCs cultured under normoxic (21% O2) and hypoxic (5% O2) conditions. Hypoxia increased protein expression of HIF (hypoxia-inducible factor)-1α and its target genes GLUT1 (glucose transporter 1) and VEGFA (vascular endothelial growth factor A) and induced mRNA and protein expressions of osteochondrogenic markers, that is, RUNX2 (runt-related transcription factor 2), SOX9 (Sry-related HMG box-9), OCN (osteocalcin) and ALP (alkaline phosphatase), and induced a time-dependent calcification of the extracellular matrix of VSMCs. HIF-1 inhibition by chetomin abrogated the effect of hypoxia on osteochondrogenic markers and abolished extracellular matrix calcification. Hypoxia triggered the production of reactive oxygen species, which was inhibited by chetomin. Scavenging reactive oxygen species by N-acetyl cysteine attenuated hypoxia-mediated upregulation of HIF-1α, RUNX2, and OCN protein expressions and inhibited extracellular matrix calcification, which effect was mimicked by a specific hydrogen peroxide scavenger sodium pyruvate and a mitochondrial reactive oxygen species inhibitor rotenone. Ex vivo culture of mice aorta under hypoxic conditions triggered calcification which was inhibited by chetomin and N-acetyl cysteine. In vivo hypoxia exposure (10% O2) increased RUNX2 mRNA levels in mice lung and the aorta. Conclusions- Hypoxia contributes to vascular calcification through the induction of osteochondrogenic differentiation of VSMCs in an HIF-1-dependent and mitochondria-derived reactive oxygen species-dependent manner.

Renal control of disease tolerance to malaria.

Malaria, the disease caused by Plasmodium spp. infection, remains a major global cause of morbidity and mortality. Host protection from malaria relies on immune-driven resistance mechanisms that kill Plasmodium However, these mechanisms are not sufficient per se to avoid the development of severe forms of disease. This is accomplished instead via the establishment of disease tolerance to malaria, a defense strategy that does not target Plasmodium directly. Here we demonstrate that the establishment of disease tolerance to malaria relies on a tissue damage-control mechanism that operates specifically in renal proximal tubule epithelial cells (RPTEC). This protective response relies on the induction of heme oxygenase-1 (HMOX1; HO-1) and ferritin H chain (FTH) via a mechanism that involves the transcription-factor nuclear-factor E2-related factor-2 (NRF2). As it accumulates in plasma and urine during the blood stage of Plasmodium infection, labile heme is detoxified in RPTEC by HO-1 and FTH, preventing the development of acute kidney injury, a clinical hallmark of severe malaria.

Oxidized hemoglobin forms contribute to NLRP3 inflammasome-driven IL-1ß production upon intravascular hemolysis.

Damage associated molecular patterns (DAMPs) are released form red blood cells (RBCs) during intravascular hemolysis (IVH). Extracellular heme, with its pro-oxidant, pro-inflammatory and cytotoxic effects, is sensed by innate immune cells through pattern recognition receptors such as toll-like receptor 4 and nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3), while free availability of heme is strictly controlled. Here we investigated the involvement of different hemoglobin (Hb) forms in hemolysis-associated inflammatory responses. We found that after IVH most of the extracellular heme molecules are localized in oxidized Hb forms. IVH was associated with caspase-1 activation and formation of mature IL-1ß in plasma and in the liver of C57BL/6 mice. We showed that ferrylHb (FHb) induces active IL-1ß production in LPS-primed macrophages in vitro and triggered intraperitoneal recruitment of neutrophils and monocytes, caspase-1 activation and active IL-1ß formation in the liver of C57BL/6 mice. NLRP3 deficiency provided a survival advantage upon IVH, without influencing the extent of RBC lysis or the accumulation of oxidized Hb forms. However, both hemolysis-induced and FHb-induced pro-inflammatory responses were largely attenuated in Nlrp3-/- mice. Taken together, FHb is a potent trigger of NLRP3 activation and production of IL-1ß in vitro and in vivo, suggesting that FHb may contribute to hemolysis-induced inflammation. Identification of RBC-derived DAMPs might allow us to develop new therapeutic approaches for hemolytic diseases.