Vascular Calcification Mechanisms: Updates and Renewed Insight into Signaling Pathways Involved in High Phosphate-Mediated Vascular Smooth Muscle Cell Calcification

© 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/Vascular calcification (VC) is associated with aging, cardiovascular and renal diseases and results in poor morbidity and increased mortality. VC occurs in patients with chronic kidney disease (CKD), a condition that is associated with high serum phosphate (Pi) and severe cardiovascular consequences. High serum Pi level is related to some pathologies which affect the behaviour of vascular cells, including platelets, endothelial cells (ECs) and smooth muscle cells (SMCs), and plays a central role in promoting VC. VC is a complex, active and cell-mediated process involving the transdifferentiation of vascular SMCs to a bone-like phenotype, systemic inflammation, decreased anti-calcific events (loss of calcification inhibitors), loss in SMC lineage markers and enhanced pro-calcific microRNAs (miRs), an increased intracellular calcium level, apoptosis, aberrant DNA damage response (DDR) and senescence of vascular SMCs. This review gives a brief overview of the current knowledge of VC mechanisms with a particular focus on Pi-induced changes in the vascular wall important in promoting calcification. In addition to reviewing the main findings, this review also sheds light on directions for future research in this area and discusses emerging pathways such as Pi-regulated intracellular calcium signaling, epigenetics, oxidative DNA damage and senescence-mediated mechanisms that may play critical, yet to be explored, regulatory and druggable roles in limiting VC.Peer reviewedFinal Published versio

Low pH up‐regulates interleukin‐6 mRNA in L6‐G8C5 rat skeletal muscle cells independent of pH sensing by SNAT2(SLC38A2) transporters

© 2021 The Authors. FASEB BioAdvances published by The Federation of American Societies for Experimental Biology. This is an open access article. https://creativecommons.org/licenses/by/4.0/Exercise is known to create a transient, but potent increase in skeletal muscle expression of potentially anti‐inflammatory myokine interleukin‐6 (IL‐6). This effect may be clinically important in managing chronic inflammatory states. It has previously been proposed that lactic acidosis following exercise promotes this IL‐6 up‐regulation, but the mechanism of this acidosis effect is unknown. Rat skeletal muscle cell line L6‐G8C5 has been used previously to model metabolic effects of acidosis, sensing low pH through the resulting inhibition of amino acid transporter SNAT2(SLC38A2). Use of ionophore ionomycin to model the rise in intracellular Ca2+ concentration occurring in contracting muscle strongly up‐regulates IL‐6 mRNA in L6‐G8C5 myotubes. This study used this model to test the hypothesis that low extracellular pH (7.1) enhances ionomycin‐induced IL‐6 mRNA up‐regulation by inhibiting SNAT2. Incubation of L6‐G8C5 myotubes for 6 h with 0.5 µM ionomycin at control pH (7.4) resulted in a 15‐fold increase in IL‐6 mRNA which was further enhanced (1.74‐fold) at pH 7.1. In contrast low pH had no significant effect on IL‐6 mRNA without ionomycin, nor on the IL‐6 mRNA increase that was induced by cyclic stretch. Even though pH 7.1 halved the transport activity of SNAT2, alternative methods of SNAT2 inhibition (JNK inhibitor SP600125; SNAT2 antagonist MeAIB; or SNAT2 silencing with siRNA) did not mimic the enhancing effect of low pH on IL‐6 mRNA. On the contrary, JNK inhibition blunted the effect of pH 7.1 with ionomycin, but had no effect at pH 7.4. It is concluded that low pH promotes Ca2+/ionomycin–induced up‐regulation of IL‐6 mRNA through a novel SNAT2‐independent JNK‐dependent pH‐sensing pathway not previously described in this skeletal muscle model.Peer reviewedFinal Published versio

Supramaximal calcium signaling triggers procoagulant platelet formation.

Procoagulant platelets promote thrombin generation during thrombosis. Platelets become procoagulant in an all-or-nothing manner. We investigated how distinct Ca2+ signaling between platelet subpopulations commits some platelets to become procoagulant, using the high-affinity Ca2+ indicator Fluo-4, which may become saturated during platelet stimulation, or low-affinity Fluo-5N, which reports only very high cytosolic Ca2+ concentrations. All activated platelets had high Fluo-4 fluorescence. However, in Fluo-5N-loaded platelets, only the procoagulant platelets had high fluorescence, indicating very high cytosolic Ca2+. This finding indicates a novel, "supramaximal" Ca2+ signal in procoagulant platelets (ie, much higher than normally considered maximal). Supramaximal Ca2+ signaling and the percentage of procoagulant platelets were inhibited by cyclosporin A, a mitochondrial permeability transition pore blocker, and Ru360, an inhibitor of the mitochondrial Ca2+ uniporter, with no effect on Fluo-4 fluorescence. In contrast, Synta-66, an Orai1 blocker, reduced Fluo-4 fluorescence but did not directly inhibit generation of the supramaximal Ca2+ signal. Our findings show a distinct pattern of Ca2+ signaling in procoagulant platelets and provide a new framework to interpret the role of platelet signaling pathways in procoagulant platelets. This requires reassessment of the role of different Ca2+ channels and may provide new targets to prevent formation of procoagulant platelets and limit thrombosis

Hepatic cysteine sulphinic acid decarboxylase depletion and defective taurine metabolism in a rat partial nephrectomy model of chronic kidney disease

© 2021, The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.Background: Taurine depletion occurs in patients with end-stage chronic kidney disease (CKD). In contrast, in the absence of CKD, plasma taurine is reported to increase following dietary L-glutamine supplementation. This study tested the hypothesis that taurine biosynthesis decreases in a rat CKD model, but is rectified by L-glutamine supplementation. Methods: CKD was induced by partial nephrectomy in male Sprague-Dawley rats, followed 2 weeks later by 2 weeks of 12% w/w L-glutamine supplemented diet (designated NxT) or control diet (NxC). Sham-operated control rats (S) received control diet. Results: Taurine concentration in plasma, liver and skeletal muscle was not depleted, but steady-state urinary taurine excretion (a measure of whole-body taurine biosynthesis) was strongly suppressed (28.3 ± 8.7 in NxC rats versus 78.5 ± 7.6 μmol/24 h in S, P < 0.05), accompanied by reduced taurine clearance (NxC 0.14 ± 0.05 versus 0.70 ± 0.11 ml/min/Kg body weight in S, P < 0.05). Hepatic expression of mRNAs encoding key enzymes of taurine biosynthesis (cysteine sulphinic acid decarboxylase (CSAD) and cysteine dioxygenase (CDO)) showed no statistically significant response to CKD (mean relative expression of CSAD and CDO in NxC versus S was 0.91 ± 0.18 and 0.87 ± 0.14 respectively). Expression of CDO protein was also unaffected. However, CSAD protein decreased strongly in NxC livers (45.0 ± 16.8% of that in S livers, P < 0.005). L-glutamine supplementation failed to rectify taurine biosynthesis or CSAD protein expression, but worsened CKD (proteinuria in NxT 12.5 ± 1.2 versus 6.7 ± 1.5 mg/24 h in NxC, P < 0.05). Conclusion: In CKD, hepatic CSAD is depleted and taurine biosynthesis impaired. This is important in view of taurine’s reported protective effect against cardio-vascular disease - the leading cause of death in human CKD.Peer reviewe

Global, regional, and national burden of disorders affecting the nervous system, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BackgroundDisorders affecting the nervous system are diverse and include neurodevelopmental disorders, late-life neurodegeneration, and newly emergent conditions, such as cognitive impairment following COVID-19. Previous publications from the Global Burden of Disease, Injuries, and Risk Factor Study estimated the burden of 15 neurological conditions in 2015 and 2016, but these analyses did not include neurodevelopmental disorders, as defined by the International Classification of Diseases (ICD)-11, or a subset of cases of congenital, neonatal, and infectious conditions that cause neurological damage. Here, we estimate nervous system health loss caused by 37 unique conditions and their associated risk factors globally, regionally, and nationally from 1990 to 2021.MethodsWe estimated mortality, prevalence, years lived with disability (YLDs), years of life lost (YLLs), and disability-adjusted life-years (DALYs), with corresponding 95% uncertainty intervals (UIs), by age and sex in 204 countries and territories, from 1990 to 2021. We included morbidity and deaths due to neurological conditions, for which health loss is directly due to damage to the CNS or peripheral nervous system. We also isolated neurological health loss from conditions for which nervous system morbidity is a consequence, but not the primary feature, including a subset of congenital conditions (ie, chromosomal anomalies and congenital birth defects), neonatal conditions (ie, jaundice, preterm birth, and sepsis), infectious diseases (ie, COVID-19, cystic echinococcosis, malaria, syphilis, and Zika virus disease), and diabetic neuropathy. By conducting a sequela-level analysis of the health outcomes for these conditions, only cases where nervous system damage occurred were included, and YLDs were recalculated to isolate the non-fatal burden directly attributable to nervous system health loss. A comorbidity correction was used to calculate total prevalence of all conditions that affect the nervous system combined.FindingsGlobally, the 37 conditions affecting the nervous system were collectively ranked as the leading group cause of DALYs in 2021 (443 million, 95% UI 378–521), affecting 3·40 billion (3·20–3·62) individuals (43·1%, 40·5–45·9 of the global population); global DALY counts attributed to these conditions increased by 18·2% (8·7–26·7) between 1990 and 2021. Age-standardised rates of deaths per 100 000 people attributed to these conditions decreased from 1990 to 2021 by 33·6% (27·6–38·8), and age-standardised rates of DALYs attributed to these conditions decreased by 27·0% (21·5–32·4). Age-standardised prevalence was almost stable, with a change of 1·5% (0·7–2·4). The ten conditions with the highest age-standardised DALYs in 2021 were stroke, neonatal encephalopathy, migraine, Alzheimer's disease and other dementias, diabetic neuropathy, meningitis, epilepsy, neurological complications due to preterm birth, autism spectrum disorder, and nervous system cancer.InterpretationAs the leading cause of overall disease burden in the world, with increasing global DALY counts, effective prevention, treatment, and rehabilitation strategies for disorders affecting the nervous system are needed

Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation

Vascular Calcification Mechanisms: Updates and Renewed Insight into Signaling Pathways Involved in High Phosphate-Mediated Vascular Smooth Muscle Cell Calcification

Vascular calcification (VC) is associated with aging, cardiovascular and renal diseases and results in poor morbidity and increased mortality. VC occurs in patients with chronic kidney disease (CKD), a condition that is associated with high serum phosphate (Pi) and severe cardiovascular consequences. High serum Pi level is related to some pathologies which affect the behaviour of vascular cells, including platelets, endothelial cells (ECs) and smooth muscle cells (SMCs), and plays a central role in promoting VC. VC is a complex, active and cell-mediated process involving the transdifferentiation of vascular SMCs to a bone-like phenotype, systemic inflammation, decreased anti-calcific events (loss of calcification inhibitors), loss in SMC lineage markers and enhanced pro-calcific microRNAs (miRs), an increased intracellular calcium level, apoptosis, aberrant DNA damage response (DDR) and senescence of vascular SMCs. This review gives a brief overview of the current knowledge of VC mechanisms with a particular focus on Pi-induced changes in the vascular wall important in promoting calcification. In addition to reviewing the main findings, this review also sheds light on directions for future research in this area and discusses emerging pathways such as Pi-regulated intracellular calcium signaling, epigenetics, oxidative DNA damage and senescence-mediated mechanisms that may play critical, yet to be explored, regulatory and druggable roles in limiting VC

Microparticles as biomarkers of early changes leading to cardiovascular disease in chronic kidney disease

Hyperphosphataemia in patients with advanced chronic kidney disease (CKD) is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of pro-coagulant endothelial microparticles (MPs), leading to a pro-thrombotic state, which may contribute to acute occlusive events. It is hypothesised that hyperphosphataemia leads to MP formation from ECs via an elevation of intracellular Pi, which directly inhibits phosphoprotein phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. Using cultured human endothelial cells (EAhy926), incubation with elevated extracellular Pi (2.5mM) led to a rise in intracellular Pi concentration within 90min. This was mediated by PiT-1/slc20a1 Pi transporters; and led to global accumulation of Tyr- and Ser-Thr phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing and release of 0.1 – 1 micron diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovandate or fluoride also yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay were significantly more pro-coagulant than particles derived from cells incubated in medium with a physiological level of Pi (1mM). These data demonstrate a mechanism of Pi-induced cellular stress and signalling which may be widely applicable in mammalian cells; and in ECs provides a novel pathological link between hyperphosphataemia, generation of MPs and thrombotic risk

High extracellular phosphate increases platelet polyphosphate content.

Platelet-derived extracellular polyphosphate (PolyP) is a major mediator of thrombosis. PolyP is a linear chain of inorganic phosphate (Pi) and is stored in platelet dense granules. Pi enters cells from the extracellular fluid through phosphate transporters and may be stored as PolyP. Here we show that high extracellular Pi concentration in vitro increases platelet PolyP content, in a manner dependent on phosphate transporters, IP6K and V-type ATPases. The increased PolyP also enhanced PolyP-dependent coagulation in platelet-rich plasma. These data suggest a mechanistic link between hyperphosphatemia, PolyP and enhanced coagulation, which may be important in pathologies such as chronic kidney disease

Evaluation of the effects of cisplatin and the cisplatin-alum mixture as adjuvants for increasing the efficacy of vaccination against Salmonella typhimurium in Balb/c mice

Background: Salmonella typhimurium (S. typhimurium) is one of the causative agents of intestinal and extraintestinal infections in humans. Symptoms of the mouse infection by this bacterium mimic typhoid fever in humans. Adjuvants are compounds that enhance the effectiveness of vaccines in combination with them. Alum as an adjuvant causes a shift towards Th2 immune and strengthens the humoral immunity responses. Cisplatin is a highly effective anti-tumor drug that stimulates immune responses by activating macrophages and other immune cells and is used in tumor immunotherapy. This study aimed to investigate the role of cisplatin and the cisplatin-alum mixture as adjuvants to increase the efficacy of vaccination against S. typhimurium in Balb/c mice. Methods: Male BALB/c mice were divided into five groups. Mice in the experimental groups received either the HKST vaccine alone or in combination with the adjuvants alum, cisplatin, or the cisplatin-alum. Mice in the negative control group received phosphate-buffered saline. All mice were immunized two times on days 0 and 14. Two weeks after the last immunization, immune responses to S. typhimurium were assessed by measuring the survival rate after challenge with a lethal dose of bacterium, bacterial load in the liver, interferon-gamma, and S. typhimurium-specific IgG1 and IgG2a production.  Results: The numbers of colonies in the spleen and liver cultures in all dilutions were significantly lower in cisplatin-vaccine, and cisplatin-alum vaccine immunized mice. The average rate of specific IgG2a was higher in the same groups compared to other groups. The survival rate in alum-vaccine, cisplatin-vaccine, and cisplatin-alum-vaccine groups was significantly higher than in the control group. The average rate of Interferon-gamma in cisplatin-vaccine and cisplatin- alum vaccine groups, was significantly higher than other groups. Conclusion: This study is the first to determine the role of administrating cisplatin and alum-cisplatin mixture on increasing the efficiency of the HKST vaccine in a mouse model. This study confirmed the role of cisplatin and cisplatin-alum mixture in increasing the efficiency of the HKST vaccine by using different experiments