Impact of Inducible Nitric Oxide Synthase Activation on Endothelial Behavior under Magnesium Deficiency.

Endothelial dysfunction is a crucial event in the early pathogenesis of cardiovascular diseases and is linked to magnesium (Mg) deficiency. Indeed, in endothelial cells, low Mg levels promote the acquisition of a pro-inflammatory and pro-atherogenic phenotype. This paper investigates the mechanisms by which Mg deficiency promotes oxidative stress and affects endothelial behavior in human umbilical vascular endothelial cells (HUVECs). Our data show that low Mg levels trigger oxidative stress initially by increasing NAPDH oxidase activity and then by upregulating the pro-oxidant thioredoxin-interacting protein TXNIP. The overproduction of reactive oxygen species (ROS) activates NF-κB, leading to its increased binding to the inducible nitric oxide synthase (iNOS) promoter, with the consequent increase in iNOS expression. The increased levels of nitric oxide (NO) generated by upregulated iNOS contribute to disrupting endothelial cell function by inhibiting growth and increasing permeability. In conclusion, we provide evidence that multiple mechanisms contribute to generate a pro-oxidant state under low-Mg conditions, ultimately affecting endothelial physiology. These data add support to the notion that adequate Mg levels play a significant role in preserving cardiovascular health and may suggest new approaches to prevent or manage cardiovascular diseases.

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.

Effects of Withania somnifera on Cortisol Levels in Stressed Human Subjects: A Systematic Review.

BACKGROUND: Withania somnifera (WS), a popular medicinal plant of the Solanaceae family, contains active ingredients with antioxidant, anti-inflammatory, immunomodulatory, and anti-stress activities. However, its precise mechanisms of action and optimal use as a supplement are not yet fully understood. The objective of this systematic review is to assess the impact of WS supplementation on cortisol levels in stressed humans by analyzing clinical trials conducted prior to May 2023. METHODS: The assessment was carried out following the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) by exploring the databases of EMBASE, PubMed, Google Scholar, CENTRAL, and Scopus. RESULTS: Of the 4788 articles identified, only 9 studies met the selection criteria. The selected studies varied in terms of design, results, formulations, dosages, and treatment duration (30-112 days), and involved subjects with varying degrees of stress. WS supplementation decreases cortisol secretion with no significant adverse effects. Nonetheless, none of the studies evaluated the potential impact of cortisol reduction on adrenal function and long-term effects. CONCLUSIONS: Brief-term supplementation with WS appears to have a stress-reducing effect in stressed individuals. However, since the long-term effects of WS supplementation are not yet fully understood, WS supplements should be used under medical supervision.

Low extracellular magnesium induces phenotypic and metabolic alterations in C2C12-derived myotubes.

Magnesium (Mg) has a pivotal role in upholding skeletal muscle health and optimizing performance. Its deficiency decreases muscle strength, and an association has been reported between Mg intake and sarcopenia. To gain a comprehensive understanding of the repercussions arising from low Mg concentrations on muscle behavior, we employed an in vitro model utilizing C2C12-derived myotubes. Myotubes cultured in low Mg show a significant reduction of thickness and a concomitant down-regulation of myosin heavy chain (MyHC), Myog and Myomixer. In parallel, myotubes shape their metabolism. Glycolysis is inhibited and beta-oxidation increases. These metabolic changes are consistent with the increase of MyHC I (slow) vs. MyHC II (fast) expression. We identified an essential player in these changes, namely nitric oxide (NO), as the increase in NO production appeared to orchestrate the observed modifications in myotube behavior and metabolism under low Mg conditions. Understanding these underlying mechanisms may pave the way for targeted interventions to ameliorate muscle-related conditions associated with Mg deficiency and contribute to enhancing overall muscle health and function.

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.

Aging and Vascular Disease: A Multidisciplinary Overview.

Vascular aging, i.e., the deterioration of the structure and function of the arteries over the life course, predicts cardiovascular events and mortality. Vascular degeneration can be recognized before becoming clinically symptomatic; therefore, its assessment allows the early identification of individuals at risk. This opens the possibility of minimizing disease progression. To review these issues, a search was completed using PubMed, MEDLINE, and Google Scholar from 2000 to date. As a network of clinicians and scientists involved in vascular medicine, we here describe the structural and functional age-dependent alterations of the arteries, the clinical tools for an early diagnosis of vascular aging, and the cellular and molecular events implicated. It emerges that more studies are necessary to identify the best strategy to quantify vascular aging, and to design proper physical activity programs, nutritional and pharmacological strategies, as well as social interventions to prevent, delay, and eventually revert the disease.

The Recurring Word in the Scientific Articles about the Role of Mg in Living Systems Is "Key".

Magnesium (Mg) is a versatile element involved in all aspects of life on our planet [...].

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 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.

Cluster-Assembled Zirconia Substrates Accelerate the Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells.

Due to their high mechanical strength and good biocompatibility, nanostructured zirconia surfaces (ns-ZrOx) are widely used for bio-applications. Through supersonic cluster beam deposition, we produced ZrOx films with controllable roughness at the nanoscale, mimicking the morphological and topographical properties of the extracellular matrix. We show that a 20 nm ns-ZrOx surface accelerates the osteogenic differentiation of human bone marrow-derived MSCs (bMSCs) by increasing the deposition of calcium in the extracellular matrix and upregulating some osteogenic differentiation markers. bMSCs seeded on 20 nm ns-ZrOx show randomly oriented actin fibers, changes in nuclear morphology, and a reduction in mitochondrial transmembrane potential when compared to the cells cultured on flat zirconia (flat-ZrO2) substrates and glass coverslips used as controls. Additionally, an increase in ROS, known to promote osteogenesis, was detected after 24 h of culture on 20 nm ns-ZrOx. All the modifications induced by the ns-ZrOx surface are rescued after the first hours of culture. We propose that ns-ZrOx-induced cytoskeletal remodeling transmits signals generated by the extracellular environment to the nucleus, with the consequent modulation of the expression of genes controlling cell fate.

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.

A potential protective role of magnesium in neuroCOVID

Several recent studies support a role of dysregulated magnesium homeostasis in COVID-19. In the present narrative review, we focus on the neurological aspects of this disease, collectively known as neuroCOVID, and we propose some mechanisms by which alterations of magnesium may contribute to the involvement of the nervous system in the context of SARS-CoV-2 infection. Further fundamental, translational, and clinical research is needed to underpin the potential relationships between altered magnesium status and neuro-COVID, with potentially novel therapeutic implications.

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.

Recommendation on an updated standardization of serum magnesium reference ranges.

PURPOSE: Serum magnesium is the most frequently used laboratory test for evaluating clinical magnesium status. Hypomagnesemia (low magnesium status), which is associated with many chronic diseases, is diagnosed using the serum magnesium reference range. Currently, no international consensus for a magnesemia normal range exists. Two independent groups designated 0.85 mmol/L (2.07 mg/dL; 1.7 mEq/L) as the low cut-off point defining hypomagnesemia. MaGNet discussions revealed differences in serum magnesium reference ranges used by members' hospitals and laboratories, presenting an urgent need for standardization. METHODS: We gathered and compared serum magnesium reference range values from our institutions, hospitals, and colleagues worldwide. RESULTS: Serum magnesium levels designating "hypomagnesemia" differ widely. Of 43 collected values, only 2 met 0.85 mmol/L as the low cut-off point to define hypomagnesemia. The remainder had lower cut-off values, which may underestimate hypomagnesemia diagnosis in hospital, clinical, and research assessments. Current serum magnesium reference ranges stem from "normal" populations, which unknowingly include persons with chronic latent magnesium deficit (CLMD). Serum magnesium levels of patients with CLMD fall within widely used "normal" ranges, but their magnesium status is too low for long-term health. The lower serum magnesium reference (0.85 mmol/L) proposed specifically prevents the inclusion of patients with CLMD. CONCLUSIONS: Widely varying serum magnesium reference ranges render our use of this important medical tool imprecise, minimizing impacts of low magnesium status or hypomagnesemia as a marker of disease risk. To appropriately diagnose, increase awareness of, and manage magnesium status, it is critical to standardize lower reference values for serum magnesium at 0.85 mmol/L (2.07 mg/dL; 1.7 mEq/L).

A Potential Interplay between HDLs and Adiponectin in Promoting Endothelial Dysfunction in Obesity.

Obesity is an epidemic public health problem that has progressively worsened in recent decades and is associated with low-grade chronic inflammation (LGCI) in metabolic tissues and an increased risk of several diseases. In particular, LGCI alters metabolism and increases cardiovascular risk by impairing endothelial function and altering the functions of adiponectin and high-density lipoproteins (HDLs). Adiponectin is an adipokine involved in regulating energy metabolism and body composition. Serum adiponectin levels are reduced in obese individuals and negatively correlate with chronic sub-clinical inflammatory markers. HDLs are a heterogeneous and complex class of lipoproteins that can be dysfunctional in obesity. Adiponectin and HDLs are strictly interdependent, and the maintenance of their interplay is essential for vascular function. Since such a complex network of interactions is still overlooked in clinical settings, this review aims to highlight the mechanisms involved in the impairment of the HDLs/adiponectin axis in obese patients to predict the risk of cardiovascular diseases and activate preventive countermeasures. Here, we provide a narrative review of the role of LGCI in altering HDLs, adiponectin and endothelial functions in obesity to encourage new studies about their synergic effects on cardiovascular health and disease.

The Presence of Blood-Brain Barrier Modulates the Response to Magnesium Salts in Human Brain Organoids.

Magnesium (Mg) is fundamental in the brain, where it regulates metabolism and neurotransmission and protects against neuroinflammation. To obtain insights into the molecular basis of Mg action in the brain, we investigated the effects of Mg in human brain organoids, a revolutionary 3D model to study neurobiology and neuropathology. In particular, brain organoids derived from human induced pluripotent stem cells were cultured in the presence or in the absence of an in vitro-generated blood-brain barrier (BBB), and then exposed to 1 or 5 mM concentrations of inorganic and organic Mg salts (Mg sulphate (MgSO4); Mg pidolate (MgPid)). We evaluated the modulation of NMDA and GABAergic receptors, and BDNF. Our data suggest that the presence of the BBB is essential for Mg to exert its effects on brain organoids, and that 5 mM of MgPid is more effective than MgSO4 in increasing the levels of GABA receptors and BDNF, and decreasing those of NMDA receptor. These results might illuminate novel pathways explaining the neuroprotective role of Mg.

From Cultured Vascular Cells to Vessels: The Cellular and Molecular Basis of Vascular Dysfunction in Space.

Life evolved on this planet under the pull of gravity, shielded from radiation by the magnetosphere and shaped by circadian rhythms due to Earth's rotation on its axis. Once living beings leave such a protective environment, adaptive responses are activated to grant survival. In view of long manned mission out of Earth's orbit, it is relevant to understand how humans adapt to space and if the responses activated might reveal detrimental in the long run. Here we review present knowledge about the effects on the vessels of various extraterrestrial factors on humans as well as in vivo and in vitro experimental models. It emerges that the vasculature activates complex adaptive responses finalized to supply oxygen and nutrients to all the tissues and to remove metabolic waste and carbon dioxide. Most studies point to oxidative stress and mitochondrial dysfunction as mediators of vascular alterations in space. Unraveling the cellular and molecular mechanisms involved in these adaptive processes might offer hints to design proper and personalized countermeasures to predict a safe future in space.

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.