Magnesium and inflammation: Advances and perspectives.

Magnesium is an essential element of life, involved in the regulation of metabolism and homeostasis of all the tissues. It also regulates immunological functions, acting on the cells of innate and adaptive immune systems. Magnesium deficiency primes phagocytes, enhances granulocyte oxidative burst, activates endothelial cells and increases the levels of cytokines, thus promoting inflammation. Consequently, a low magnesium status, which is often underdiagnosed, potentiates the reactivity to various immune challenges and is implicated in the pathophysiology of many common chronic diseases. Here we summarize recent advances supporting the link between magnesium deficiency, inflammatory responses and diseases, and offer new hints towards a better understanding of the underlying mechanisms.

Human endothelial cells in high glucose: New clues from culture in 3D microfluidic chips.

Several studies have demonstrated the role of high glucose in promoting endothelial dysfunction utilizing traditional two-dimensional (2D) culture systems, which, however, do not replicate the complex organization of the endothelium within a vessel constantly exposed to flow. Here we describe the response to high glucose of micro- and macro-vascular human endothelial cells (EC) cultured in biomimetic microchannels fabricated through soft lithography and perfused to generate shear stress. In 3D macrovascular EC exposed to a shear stress of 0.4 Pa respond to high glucose with cytoskeletal remodeling and alterations in cell shape. Under the same experimental conditions, these effects are more pronounced in microvascular cells that show massive cytoskeletal disassembly and apoptosis after culture in high glucose. However, when exposed to a shear stress of 4 Pa, which is physiological in the microvasculature, human dermal microvascular endothelial cells (HDMEC) show alterations of the cytoskeleton but no apoptosis. This result emphasizes the sensitivity of HDMEC to different regimens of flow. No significant variations in the thickness of glycocalyx were detected in both human endothelial cells from the umbilical vein and HDMEC exposed to high glucose in 3D, whereas clear differences emerge between cells cultured in static 2D versus microfluidic channels. We conclude that culture in microfluidic microchannels unveils unique insights into endothelial dysfunction by high glucose.

Long-term kidney outcome of patients with rheumatological diseases and antineutrophil cytoplasmic antibody-glomerulonephritis: comparison with a primitive ANCA-glomerulonephritis cohort.

OBJECTIVES: Antineutrophil cytoplasmic antibody (ANCA) may appear in the course of rheumatic diseases (RD) but the kidney involvement is very rare and the prognosis poorly defined. METHODS: We retrospectively identified patients with RD among 153 patients with ANCA glomerulonephritis (ANCA-GN). Their clinical/histological presentation and outcome were compared with that of primitive ANCA-GN patients (1:4) matched for sex, age, ANCA type and follow-up. RESULTS: Nine patients (5.9%) were included: three had rheumatoid arthritis, two systemic sclerosis, two psoriatic arthritis, one ankylosing spondylitis and one seronegative spondylarthritis. Seven patients were MPO positive, two PR3 positive. ANCA-GN developed 74 months after RD with microscopic haematuria and acute kidney dysfunction in all but two patients. After 68-month follow-up, four patients (44.4%) achieved response to therapy defined as eGFR >60/min/1,73 m2 or stable, no microscopic haematuria and negative ANCA. At ANCA-GN diagnosis, serum creatinine and C-reactive protein were significantly lower in RD-ANCA-GN (2.38 vs. 3.34mg/dl, p=0.05 and 2.3mg/dl vs. 7.2mg/dl; p=0.05, respectively) while haemoglobin was higher (12.3g/dl vs. 9.3g/dl p<0.01) than in the 36 primitive ANCA-GN patients of control group. At kidney biopsy, focal forms were more frequent in RD patients (44.45% vs. 18.75%, p=0.11). The treatment between the two groups was not significantly different. At last observation, the percentage of patients with ESKD was lower in RD than in controls (11.1%vs. 30.5%; p=0.23). CONCLUSIONS: Patients with RD seem to develop ANCA-GN with less severe clinical/histological kidney involvement, and better long-term kidney survival than primitive ANCA-GN. This is probably due to the strict monitoring of RD patients that allows a prompter ANCA-GN diagnosis and treatment.

The Role of Txnip in Mediating Low-Magnesium-Driven Endothelial Dysfunction.

Magnesium deficiency is associated with a greater risk of developing cardiovascular diseases since this cation is fundamental in regulating vascular function. This clinical evidence is sustained by in vitro studies showing that culturing endothelial cells in low concentrations of magnesium promotes the acquisition of a pro-oxidant and pro-inflammatory phenotype. Here, we show that the increase in reactive oxygen species in endothelial cells in low-magnesium-containing medium is due to the upregulation of the pro-oxidant protein thioredoxin interacting protein (TXNIP), with a consequent accumulation of lipid droplets and increase in endothelial permeability through the downregulation and relocalization of junctional proteins. Silencing TXNIP restores the endothelial barrier and lipid content. Because (i) mitochondria serve multiple roles in shaping cell function, health and survival and (ii) mitochondria are the main intracellular stores of magnesium, it is of note that no significant alterations were detected in their morphology and dynamics in our experimental model. We conclude that TXNIP upregulation contributes to low-magnesium-induced endothelial dysfunction in vitro.

The Effects of Sirolimus and Magnesium on Primary Human Coronary Endothelial Cells: An In Vitro Study.

Drug eluting magnesium (Mg) bioresorbable scaffolds represent a novel paradigm in percutaneous coronary intervention because Mg-based alloys are biocompatible, have adequate mechanical properties and can be resorbed without adverse events. Importantly, Mg is fundamental in many biological processes, mitigates the inflammatory response and is beneficial for the endothelium. Sirolimus is widely used as an antiproliferative agent in drug eluting stents to inhibit the proliferation of smooth muscle cells, thus reducing the occurrence of stent restenosis. Little is known about the potential interplay between sirolimus and Mg in cultured human coronary artery endothelial cells (hCAEC). Therefore, the cells were treated with sirolimus in the presence of different concentrations of extracellular Mg. Cell viability, migration, barrier function, adhesivity and nitric oxide synthesis were assessed. Sirolimus impairs the viability of subconfluent, but not of confluent cells independently from the concentration of Mg in the culture medium. In confluent cells, sirolimus inhibits migration, while it cooperates with Mg in exerting an anti-inflammatory action that might have a role in preventing restenosis and thrombosis.

The presence of BBB hastens neuronal differentiation of cerebral organoids - The potential role of endothelial derived BDNF.

Despite remaining the best in vitro model to resemble the human brain, a weakness of human cerebral organoids is the lack of the endothelial component that in vivo organizes in the blood brain barrier (BBB). Since the BBB is crucial to control the microenvironment of the nervous system, this study proposes a co-culture of BBB and cerebral organoids. We utilized a BBB model consisting of primary human brain microvascular endothelial cells and astrocytes in a transwell system. Starting from induced Pluripotent Stem Cells (iPSCs) we generated human cerebral organoids which were then cultured in the absence or presence of an in vitro model of BBB to evaluate potential effects on the maturation of cerebral organoids. By morphological analysis, it emerges that in the presence of the BBB the cerebral organoids are better organized than controls in the absence of the BBB. This effect might be due to Brain Derived Neurotrophic Factor (BDNF), a neurotrophic factor released by the endothelial component of the BBB, which is involved in neurodevelopment, neuroplasticity and neurosurvival.

Magnesium and the Brain: A Focus on Neuroinflammation and Neurodegeneration.

Magnesium (Mg) is involved in the regulation of metabolism and in the maintenance of the homeostasis of all the tissues, including the brain, where it harmonizes nerve signal transmission and preserves the integrity of the blood-brain barrier. Mg deficiency contributes to systemic low-grade inflammation, the common denominator of most diseases. In particular, neuroinflammation is the hallmark of neurodegenerative disorders. Starting from a rapid overview on the role of magnesium in the brain, this narrative review provides evidences linking the derangement of magnesium balance with multiple sclerosis, Alzheimer's, and Parkinson's diseases.

Magnesium Homeostasis in Myogenic Differentiation-A Focus on the Regulation of TRPM7, MagT1 and SLC41A1 Transporters.

Magnesium (Mg) is essential for skeletal muscle health, but little is known about the modulation of Mg and its transporters in myogenic differentiation. Here, we show in C2C12 murine myoblasts that Mg concentration fluctuates during their differentiation to myotubes, declining early in the process and reverting to basal levels once the cells are differentiated. The level of the Mg transporter MagT1 decreases at early time points and is restored at the end of the process, suggesting a possible role in the regulation of intracellular Mg concentration. In contrast, TRPM7 is rapidly downregulated and remains undetectable in myotubes. The reduced amounts of TRPM7 and MagT1 are due to autophagy, one of the proteolytic systems activated during myogenesis and essential for the membrane fusion process. Moreover, we investigated the levels of SLC41A1, which increase once cells are differentiated, mainly through transcriptional regulation. In conclusion, myogenesis is associated with alterations of Mg homeostasis finely tuned through the modulation of MagT1, TRPM7 and SLC41A1.

A Comparison of Doxorubicin-Resistant Colon Cancer LoVo and Leukemia HL60 Cells: Common Features, Different Underlying Mechanisms.

Chemoresistance causes cancer relapse and metastasis, thus remaining the major obstacle to cancer therapy. While some light has been shed on the underlying mechanisms, it is clear that chemoresistance is a multifaceted problem strictly interconnected with the high heterogeneity of neoplastic cells. We utilized two different human cell lines, i.e., LoVo colon cancer and promyelocytic leukemia HL60 cells sensitive and resistant to doxorubicin (DXR), largely used as a chemotherapeutic and frequently leading to chemoresistance. LoVo and HL60 resistant cells accumulate less reactive oxygen species by differently modulating the levels of some pro- and antioxidant proteins. Moreover, the content of intracellular magnesium, known to contribute to protect cells from oxidative stress, is increased in DXR-resistant LoVo through the upregulation of MagT1 and in DXR-resistant HL60 because of the overexpression of TRPM7. In addition, while no major differences in mitochondrial mass are observed in resistant HL60 and LoVo cells, fragmented mitochondria due to increased fission and decreased fusion are detected only in resistant LoVo cells. We conclude that DXR-resistant cells evolve adaptive mechanisms to survive DXR cytotoxicity by activating different molecular pathways.

Mitophagy contributes to endothelial adaptation to simulated microgravity.

Exposure to real or simulated microgravity is sensed as a stress by mammalian cells, which activate a complex adaptive response. In human primary endothelial cells, we have recently shown the sequential intervention of various stress proteins which are crucial to prevent apoptosis and maintain cell function. We here demonstrate that mitophagy contributes to endothelial adaptation to gravitational unloading. After 4 and 10 d of exposure to simulated microgravity in the rotating wall vessel, the amount of BCL2 interacting protein 3, a marker of mitophagy, is increased and, in parallel, mitochondrial content, oxygen consumption, and maximal respiratory capacity are reduced, suggesting the acquisition of a thrifty phenotype to meet the novel metabolic challenges generated by gravitational unloading. Moreover, we suggest that microgravity induced-disorganization of the actin cytoskeleton triggers mitophagy, thus creating a connection between cytoskeletal dynamics and mitochondrial content upon gravitational unloading.

Magnesium Deficiency Induces Lipid Accumulation in Vascular Endothelial Cells via Oxidative Stress-The Potential Contribution of EDF-1 and PPARγ.

BACKGROUND: Magnesium deficiency contributes to atherogenesis partly by promoting the dysfunction of endothelial cells, which are critical in vascular homeostasis and diseases. Since EDF-1 and PPARγ regulate crucial endothelial activities, we investigated the modulation of these proteins involved in lipogenesis as well the deposition of lipids in human endothelial cells cultured in different concentrations of magnesium. METHODS: Human endothelial cells from the umbilical vein were cultured in medium containing from 0.1 to 5 mM magnesium for 24 h. The levels of EDF-1 and PPARγ were visualized by Western blot. Reactive oxygen species (ROS) were measured by DCFDA. Lipids were detected after O Red Oil staining. RESULTS: Magnesium deficiency leads to the accumulation of ROS which upregulate EDF-1. Further, PPARγ is increased after culture in low magnesium, but independently from ROS. Moreover, lipids accumulate in magnesium-deficient cells. CONCLUSIONS: Our results suggest that magnesium deficiency leads to the deposition of lipids by inducing EDF-1 and PPARγ. The increase in intracellular lipids might be interpreted as an adaptive response of endothelial cells to magnesium deficiency.

The dynamic adaptation of primary human endothelial cells to simulated microgravity.

Culture of human endothelial cells for 10 d in real microgravity onboard the International Space Station modulated more than 1000 genes, some of which are involved in stress response. On Earth, 24 h after exposure to simulated microgravity, endothelial cells up-regulate heat shock protein (HSP) 70. To capture a broad view of endothelial stress response to gravitational unloading, we cultured primary human endothelial cells for 4 and 10 d in the rotating wall vessel, a U.S. National Aeronautics and Space Administration-developed surrogate system for benchtop microgravity research on Earth. We highlight the crucial role of the early increase of HSP70 because its silencing markedly impairs cell survival. Once HSP70 up-regulation fades away after 4 d of simulated microgravity, a complex and articulated increase of various stress proteins (sirtuin 2, paraoxonase 2, superoxide dismutase 2, p21, HSP27, and phosphorylated HSP27, all endowed with cytoprotective properties) occurs and counterbalances the up-regulation of the pro-oxidant thioredoxin interacting protein (TXNIP). Interestingly, TXNIP was the most overexpressed transcript in endothelial cells after spaceflight. We conclude that HSP70 up-regulation sustains the initial adaptive response of endothelial cells to mechanical unloading and drives them toward the acquisition of a novel phenotype that maintains cell viability and function through the sequential involvement of different stress proteins.-Cazzaniga, A., Locatelli, L., Castiglioni, S., Maier, J. A. M. The dynamic adaptation of primary human endothelial cells to simulated microgravity.

The simultaneous downregulation of TRPM7 and MagT1 in human mesenchymal stem cells in vitro: Effects on growth and osteogenic differentiation.

The magnesium transporters TRPM7 and MagT1 are overexpressed in osteoblastogenesis. We have shown that silencing either TRPM7 or MagT1 accelerates the osteogenic differentiation of human bone mesenchymal stem cells. Here we demonstrate that the simultaneous downregulation of TRPM7 and MagT1 inhibits cell growth and activates autophagy, which is required in the early phases of osteoblastogenesis. In TRPM7/MagT1 downregulating cells the expression of two transcription factors required for activating osteogenesis, i.e. RUNX2 and OSTERIX, is induced more than in the controls both in the presence and in the absence of osteogenic stimuli, while COL1A1 is upregulated in co-silencing cells as much as in the controls. This explains why we found no differences in calcium deposition. We conclude that one of the two transporters should be expressed to accelerate osteogenic differentiation.

The Contribution of EDF1 to PPARγ Transcriptional Activation in VEGF-Treated Human Endothelial Cells.

Vascular endothelial growth factor (VEGF) is important for maintaining healthy endothelium, which is crucial for vascular integrity. In this paper, we show that VEGF stimulates the nuclear translocation of endothelial differentiation-related factor 1 (EDF1), a highly conserved intracellular protein implicated in molecular events that are pivotal to endothelial function. In the nucleus, EDF1 serves as a transcriptional coactivator of peroxisome proliferator-activated receptor gamma (PPARγ), which has a protective role in the vasculature. Indeed, silencing EDF1 prevents VEGF induction of PPARγ activity as detected by gene reporter assay. Accordingly, silencing EDF1 markedly inhibits the stimulatory effect of VEGF on the expression of FABP4, a PPARγ-inducible gene. As nitric oxide is a marker of endothelial function, it is noteworthy that we report a link between EDF1 silencing, decreased levels of FABP4, and nitric oxide production. We conclude that EDF1 is required for VEGF-induced activation of the transcriptional activity of PPARγ.

Cervical spine malformation in cornelia de lange syndrome: a report of three patients.

Cornelia de Lange syndrome (CdLS) is a complex genetic disease with skeletal involvement mostly related to upper limb malformations. We report on three males with clinical and molecular diagnoses of CdLS. Besides typical CdLS features, all showed different cervical spine malformations. To the best of our knowledge, this is an unusual malformation in the CdLS phenotypic spectrum.

Unlocking endothelial barrier restoration: FX06 in systemic capillary leak syndrome and beyond.

Increased vascular permeability is a prevalent feature in a wide spectrum of clinical conditions, but no effective treatments to restore the endothelial barrier are available. Idiopathic systemic capillary leak syndrome (ISCLS) is a life-threatening Paroxysmal Permeability Disorder characterized by abrupt, massive plasma extravasation. This condition serves as a robust model for investigating therapeutic approaches targeting interendothelial junctions. We conducted a single-center, interventional in vitro study at the Referral Center for ISCLS in Italy, involving four diagnosed ISCLS patients, aiming at investigating the effects of FX06, a Bß15-42 fibrin-derived peptide binding to VE-Cadherin, on endothelial barrier exposed to intercritical and acute ISCLS sera. The Transwell Permeability Assay was used to assess the permeability of human umbilical vein endothelial cells (HUVECs) exposed to ISCLS sera with or without FX06 (50 µg/ml). Acute ISCLS serum was also tested in a three-dimensional microfluidic device. Nitric oxide (NO), VE-Cadherin localization, and cytoskeletal organization were also assessed. In two and three-dimensional systems, ISCLS sera increased endothelial permeability, with a more pronounced effect for acute sera. Furthermore, acute sera altered VE-Cadherin localization and cytoskeletal organization. NO levels remained unchanged. FX06 restored the endothelial barrier function by influencing cellular localization rather than VE-Cadherin levels. In conclusion, FX06 prevents and reverts the hyperpermeability induced by ISCLS sera. These preliminary yet promising results provide initial evidence of the in vitro efficacy of a drug targeting the underlying pathophysiological mechanisms of ISCLS. Moreover, this approach may hold potential for addressing hyperpermeability in a spectrum of clinical conditions beyond ISCLS.

The value of the current histological scores and classifications of ANCA glomerulonephritis in predicting long-term outcome.

Background: Three different histological scores-histopathologic classification (Berden), Renal Risk Score (RRS) and the Mayo Clinic Chronicity Score (MCCS)-for anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis (ANCA-GN) were compared to evaluate their association with patient and kidney prognosis of ANCA-GN. Methods: Patients aged >18 years with at least 1 year of follow-up and biopsy-proven ANCA-GN entered this retrospective study. Renal biopsies were classified according to Berden's classification, RRS and MCCS. The first endpoint was end-stage kidney disease (ESKD), defined as chronic dialysis or estimated glomerular filtration rate <15 mL/min/1.73 m2. The second endpoint was ESKD or death. Results: Of 152 patients 84 were males, with median age of 63.8 years and followed for 46.9 (interquartile range 12.8-119) months, 59 (38.8%) reached the first endpoint and 20 died. The Kaplan-Meier curves showed that Berden and RRS were associated with first (Berden: P = .004, RRS: P < .001) and second (Berden: P = .001, RRS: P < .001) endpoint, MCCS with the first endpoint only when minimal + mild vs moderate + severe groups were compared (P = .017), and with the second endpoint (P < .001). Among the clinical/histological presentation features, arterial hypertension [odds ratio (OR) = 2.75, confidence interval (95% CI) 1.50-5.06; P = .0011], serum creatinine (OR = 1.17, 95% CI 1.09-1.25; P < .0001), and the percentage of normal glomeruli (OR = 0.97, 95% CI 0.96-0.99; P = .009) were the independent predictors of ESKD at multivariate analysis. When the three scores were included in multivariate analysis, RRS (OR = 2.21, 95% CI 1.15-4.24; P = .017) and MCCS (OR = 2.03, 95% CI 1.04-3.95; P = .037) remained predictive of ESKD, but Berden (OR = 1.17, 95% CI 0.62-2.22; P = .691) did not. Conclusion: RRS and MCCS scores were independent predictors of kidney survival together with high serum creatinine and arterial hypertension at diagnosis, while Berden classification was not.

A boy with Burkitt lymphoma associated with Noonan syndrome due to a mutation in RAF1.

This article reports on an association between Burkitt lymphoma and Noonan syndrome (NS) due to a RAF1 gene mutation. The patient was a 7-year-old boy with NS, who was included in the first series reporting the association between Noonan and RAF1, and who later presented with a 2-week history of asymptomatic unilateral tonsillar swelling and ipsilateral cervical lymphadenopathy. Histological and biological examinations of the tonsillar biopsy led to the diagnosis of Burkitt lymphoma. While there is a well-established association between NS and solid cell tumors, this is the first case described in the literature of Burkitt lymphoma in a patient with NS, and adds to the growing list of data supporting neoplasia's association with NS.

The Interplay between TRPM7 and MagT1 in Maintaining Endothelial Magnesium Homeostasis.

The transient receptor potential cation channel subfamily M member 7 (TRPM7) is an ubiquitous channel fused to an α-kinase domain involved in magnesium (Mg) transport, and its level of expression has been proposed as a marker of endothelial function. To broaden our present knowledge about the role of TRPM7 in endothelial cells, we generated stable transfected Human Endothelial Cells derived from the Umbilical Vein (HUVEC). TRPM7-silencing HUVEC maintain the actin fibers' organization and mitochondrial network. They produce reduced amounts of reactive oxygen species and grow faster than controls. Intracellular Mg concentration does not change in TRPM7-silencing or -expressing HUVEC, while some differences emerged when we analyzed intracellular Mg distribution. While the levels of the plasma membrane Mg transporter Solute Carrier family 41 member 1 (SLC41A1) and the mitochondrial channel Mrs2 remain unchanged, the highly selective Magnesium Transporter 1 (MagT1) is upregulated in TRPM7-silencing HUVEC through transcriptional regulation. We propose that the increased amounts of MagT1 grant the maintenance of intracellular Mg concentrations when TRPM7 is not expressed in endothelial cells.

Ultrastructural features mirror metabolic derangement in human endothelial cells exposed to high glucose.

High glucose-induced endothelial dysfunction is the early event that initiates diabetes-induced vascular disease. Here we employed Cryo Soft X-ray Tomography to obtain three-dimensional maps of high D-glucose-treated endothelial cells and their controls at nanometric spatial resolution. We then correlated ultrastructural differences with metabolic rewiring. While the total mitochondrial mass does not change, high D-glucose promotes mitochondrial fragmentation, as confirmed by the modulation of fission-fusion markers, and dysfunction, as demonstrated by the drop of membrane potential, the decreased oxygen consumption and the increased production of reactive oxygen species. The 3D ultrastructural analysis also indicates the accumulation of lipid droplets in cells cultured in high D-glucose. Indeed, because of the decrease of fatty acid ß-oxidation induced by high D-glucose concentration, triglycerides are esterified into fatty acids and then stored into lipid droplets. We propose that the increase of lipid droplets represents an adaptive mechanism to cope with the overload of glucose and associated oxidative stress and metabolic dysregulation.