Molecular mechanisms underlying TNFα-induced mitochondrial fragmentation in human airway smooth muscle cells.

Tumor necrosis factor α (TNFα), a proinflammatory cytokine, plays a significant role in mediating the effects of acute inflammation in response to allergens, pollutants, and respiratory infections. Previously, we showed that acute exposure to TNFα induces mitochondrial fragmentation in human airway smooth muscle (hASM) cells, which is associated with increased expression of dynamin-related protein 1 (DRP1). Phosphorylation of DRP1 at serine 616 (pDRP1S616) promotes its translocation and binding to the outer mitochondrial membrane (OMM) and mediates mitochondrial fragmentation. Previously, we reported that TNFα exposure triggers protein unfolding and triggers an endoplasmic reticulum (ER) stress response involving phosphorylation of inositol-requiring enzyme 1α (pIRE1α) at serine 724 (pIRE1αS724) and subsequent splicing of X-box binding protein 1 (XBP1s) in hASM cells. We hypothesize that TNFα-mediated activation of the pIRE1αS724/XBP1s ER stress pathway in hASM cells transcriptionally activates genes that encode kinases responsible for pDRP1S616 phosphorylation. Using 3-D confocal imaging of MitoTracker green-labeled mitochondria, we found that TNFα treatment for 6 h induces mitochondrial fragmentation in hASM cells. We also confirmed that 6 h TNFα treatment activates the pIRE1α/XBP1s ER stress pathway. Using in silico analysis and ChIP assay, we showed that CDK1 and CDK5, kinases involved in the phosphorylation of pDRP1S616, are transcriptionally targeted by XBP1s. TNFα treatment increased the binding affinity of XBP1s on the promoter regions of CDK1 and CDK5, and this was associated with an increase in pDRP1S616 and mitochondria fragmentation. This study reveals a new underlying molecular mechanism for TNFα-induced mitochondrial fragmentation in hASM cells.NEW & NOTEWORTHY Airway inflammation is increasing worldwide. Proinflammatory cytokines mediate an adaptive mechanism to overcome inflammation-induced cellular stress. Previously, we reported that TNFα mediates hASM cellular responses, leading to increased force and ATP consumption associated with increased O2 consumption, and oxidative stress. This study indicates that TNFα induces ER stress, which induces mitochondrial fragmentation via pIRE1αS724/XBP1s mediated CDK1/5 upregulation and pDRP1S616 phosphorylation. Mitochondrial fragmentation may promote hASM mitochondrial biogenesis to maintain healthy mitochondrial pool.

Management of patients with hypertension and chronic kidney disease referred to Hypertension Excellence Centres among 27 countries. On behalf of the European Society of Hypertension Working Group on Hypertension and the Kidney.

Objective Real-life management of patients with hypertension and chronic kidney disease (CKD) among European Society of Hypertension Excellence Centres (ESH-ECs) is unclear : we aimed to investigate it. Methods A survey was conducted in 2023. The questionnaire contained 64 questions asking ESH-ECs representatives to estimate how patients with CKD are managed. Results Overall, 88 ESH-ECS representatives from 27 countries participated. According to the responders, renin-angiotensin system (RAS) blockers, calcium-channel blockers and thiazides were often added when these medications were lacking in CKD patients, but physicians were more prone to initiate RAS blockers (90% [interquartile range: 70-95%]) than MRA (20% [10-30%]), SGLT2i (30% [20-50%]) or (GLP1-RA (10% [5-15%]). Despite treatment optimisation, 30% of responders indicated that hypertension remained uncontrolled (30% (15-40%) vs 18% [10%-25%]) in CKD and CKD patients, respectively). Hyperkalemia was the most frequent barrier to initiate RAS blockers, and dosage reduction was considered in 45% of responders when kalaemia was 5.5-5.9 mmol/L. Conclusions RAS blockers are initiated in most ESH-ECS in CKD patients, but MRA and SGLT2i initiations are less frequent. Hyperkalemia was the main barrier for initiation or adequate dosing of RAS blockade, and RAS blockers' dosage reduction was the usual management.

What is the context? Hypertension is a strong independent risk factor for development of chronic kidney disease (CKD) and progression of CKD to ESKD. Improved adherence to the guidelines in the treatment of CKD is believed to provide further reduction of cardiorenal events. European Society of Hypertension Excellence Centres (ESH-ECs) have been developed in Europe to provide excellency regarding management of patients with hypertension and implement guidelines. Numerous deficits regarding general practitioner CKD screening, use of nephroprotective drugs and referral to nephrologists prior to referral to ESH-ECs have been reported. In contrast, real-life management of these patients among ESH-ECs is unknown. Before implementation of strategies to improve guideline adherence in Europe, we aimed to investigate how patients with CKD are managed among the ESH-ECs.What is the study about? In this study, a survey was conducted in 2023 by the ESH to assess management of CKD patients referred to ESH-ECs. The questionnaire contained 64 questions asking ESH-ECs representatives to estimate how patients with CKD are managed among their centres.What are the results? RAAS blockers are initiated in 90% of ESH-ECs in CKD patients, but the initiation of MRA and SGLT2i is less frequently done. Hyperkalemia is the main barrier for initiation or adequate dosing of RAAS blockade, and its most reported management was RAAS blockers dosage reduction. These findings will be crucial to implement strategies in order to improve management of patients with CKD and guideline adherence among ESH-ECs.

Molecular Mechanisms Underlying TNFα-Induced Mitochondrial Biogenesis in Human Airway Smooth Muscle.

Proinflammatory cytokines such as TNFα mediate airway inflammation. Previously, we showed that TNFα increases mitochondrial biogenesis in human ASM (hASM) cells, which is associated with increased PGC1α expression. We hypothesized that TNFα induces CREB and ATF1 phosphorylation (pCREBS133 and pATF1S63), which transcriptionally co-activate PGC1α expression. Primary hASM cells were dissociated from bronchiolar tissue obtained from patients undergoing lung resection, cultured (one-three passages), and then differentiated by serum deprivation (48 h). hASM cells from the same patient were divided into two groups: TNFα (20 ng/mL) treated for 6 h and untreated controls. Mitochondria were labeled using MitoTracker green and imaged using 3D confocal microscopy to determine mitochondrial volume density. Mitochondrial biogenesis was assessed based on relative mitochondrial DNA (mtDNA) copy number determined by quantitative real-time PCR (qPCR). Gene and/or protein expression of pCREBS133, pATF1S63, PCG1α, and downstream signaling molecules (NRFs, TFAM) that regulate transcription and replication of the mitochondrial genome, were determined by qPCR and/or Western blot. TNFα increased mitochondrial volume density and mitochondrial biogenesis in hASM cells, which was associated with an increase in pCREBS133, pATF1S63 and PCG1α expression, with downstream transcriptional activation of NRF1, NRF2, and TFAM. We conclude that TNFα increases mitochondrial volume density in hASM cells via a pCREBS133/pATF1S63/PCG1α-mediated pathway.

Chemical Chaperone 4-PBA Mitigates Tumor Necrosis Factor Alpha-Induced Endoplasmic Reticulum Stress in Human Airway Smooth Muscle.

Airway inflammation and pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) underlie the pathophysiology of respiratory diseases, including asthma. Previously, we showed that TNFα activates the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 spliced (XBP1s) endoplasmic reticulum (ER) stress pathway in human airway smooth muscle (hASM) cells. The ER stress pathway is activated by the accumulation of unfolded proteins in the ER. Accordingly, chemical chaperones such as 4-phenylbutyric acid (4-PBA) may reduce ER stress activation. In the present study, we hypothesized that chemical chaperone 4-PBA mitigates TNFα-induced ER stress in hASM cells. hASM cells were isolated from bronchiolar tissue obtained from five patients with no history of smoking or respiratory diseases. The hASM cells' phenotype was confirmed via the expression of alpha-smooth muscle actin and elongated morphology. hASM cells from the same patient sample were then separated into three 12 h treatment groups: (1) TNFα (20 ng/mL), (2) TNFα + 4-PBA (1 µM, 30 min pretreatment), and (3) untreated control. The expressions of total IRE1α and phosphorylated IRE1α (pIRE1αS724) were determined through Western blotting. The splicing of XBP1 mRNA was analyzed using RT-PCR. We found that TNFα induced an increase in pIRE1αS724 phosphorylation, which was mitigated by treatment with chemical chaperone 4-PBA. We also found that TNFα induced an increase in XBP1s mRNA, which was also mitigated by treatment with chemical chaperone 4-PBA. These results support our hypothesis and indicate that chemical chaperone 4-PBA treatment mitigates TNFα-induced ER stress in hASM cells.

Cell-Based Measurement of Mitochondrial Function in Human Airway Smooth Muscle Cells.

Cellular mitochondrial function can be assessed using high-resolution respirometry that measures the O2 consumption rate (OCR) across a number of cells. However, a direct measurement of cellular mitochondrial function provides valuable information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, which participates in both the tricarboxylic acid (TCA) cycle and electron transport chain (ETC) as Complex II. In this study, we determine the maximum velocity of the SDH reaction (SDHmax) in individual human airway smooth muscle (hASM) cells. To measure SDHmax, hASM cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT, reaction indicator). As the reaction proceeded, the change in optical density (OD) due to the reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) was measured using a confocal microscope with the pathlength for light absorbance tightly controlled. SDHmax was determined during the linear period of the SDH reaction and expressed as mmol fumarate/liter of cell/min. We determine that this technique is rigorous and reproducible, and reliable for the measurement of mitochondrial function in individual cells.

TNFα induces mitochondrial fragmentation and biogenesis in human airway smooth muscle.

In human airway smooth muscle (hASM), mitochondrial volume density is greater in asthmatic patients compared with normal controls. There is also an increase in mitochondrial fragmentation in hASM of moderate asthmatics associated with an increase in dynamin-related protein 1 (Drp1) and a decrease in mitofusin 2 (Mfn2) expression, mitochondrial fission, and fusion proteins, respectively. Proinflammatory cytokines such TNFα contribute to hASM hyperreactivity and cell proliferation associated with asthma. However, the involvement of proinflammatory cytokines in mitochondrial remodeling is not clearly established. In nonasthmatic hASM cells, mitochondria were labeled using MitoTracker Red and imaged in three dimensions using a confocal microscope. After 24-h TNFα exposure, mitochondria in hASM cells were more fragmented, evidenced by decreased form factor and aspect ratio and increased sphericity. Associated with increased mitochondrial fragmentation, Drp1 expression increased while Mfn2 expression was reduced. TNFα also increased mitochondrial biogenesis in hASM cells reflected by increased peroxisome proliferator-activated receptor-γ coactivator 1α expression and increased mitochondrial DNA copy number. Associated with mitochondrial biogenesis, TNFα exposure also increased mitochondrial volume density and porin expression, resulting in an increase in maximum O2 consumption rate. However, when normalized for mitochondrial volume density, O2 consumption rate per mitochondrion was reduced by TNFα exposure. Associated with mitochondrial fragmentation and biogenesis, TNFα also increased hASM cell proliferation, an effect mimicked by siRNA knockdown of Mfn2 expression and mitigated by Mfn2 overexpression. The results of this study support our hypothesis that in hASM cells exposed to TNFα mitochondria are more fragmented, with an increase in mitochondrial biogenesis and mitochondrial volume density resulting in reduced O2 consumption rate per mitochondrion.

Dynamic cytosolic Ca2+ and force responses to muscarinic stimulation in airway smooth muscle.

During agonist stimulation of airway smooth muscle (ASM), agonists such as ACh induce a transient increase in cytosolic Ca2+ concentration ([Ca2+]cyt), which leads to a contractile response [excitation-contraction (E-C) coupling]. Previously, the sensitivity of the contractile response of ASM to elevated [Ca2+]cyt (Ca2+ sensitivity) was assessed as the ratio of maximum force to maximum [Ca2+]cyt. However, this static assessment of Ca2+ sensitivity overlooks the dynamic nature of E-C coupling in ASM. In this study, we simultaneously measured [Ca2+]cyt and isometric force responses to three concentrations of ACh (1, 2.6, and 10 µM). Both maximum [Ca2+]cyt and maximum force responses were ACh concentration dependent, but force increased disproportionately, thereby increasing static Ca2+ sensitivity. The dynamic properties of E-C coupling were assessed in several ways. The temporal delay between the onset of ACh-induced [Ca2+]cyt and onset force responses was not affected by ACh concentration. The rates of rise of the ACh-induced [Ca2+]cyt and force responses increased with increasing ACh concentration. The integral of the phase-loop plot of [Ca2+]cyt and force from onset to steady state also increased with increasing ACh concentration, whereas the rate of relaxation remained unchanged. Although these results suggest an ACh concentration-dependent increase in the rate of cross-bridge recruitment and in the rate of rise of [Ca2+]cyt, the extent of regulatory myosin light-chain (rMLC20) phosphorylation was not dependent on ACh concentration. We conclude that the dynamic properties of [Ca2+]cyt and force responses in ASM are dependent on ACh concentration but reflect more than changes in the extent of rMLC20 phosphorylation.

Extramyocellular interleukin-6 influences skeletal muscle mitochondrial physiology through canonical JAK/STAT signaling pathways.

Interleukin-6 (IL-6) is a pleiotropic cytokine that has been shown to be produced acutely by skeletal muscle in response to exercise, yet chronically elevated with obesity and aging. The mechanisms by which IL-6 influences skeletal muscle mitochondria acutely and chronically are unclear. To better understand the influence of extramyocellular IL-6 on skeletal muscle mitochondrial physiology, we treated differentiated myotubes with exogenous IL-6 to evaluate the dose- and duration-dependent effects of IL-6 on salient aspects of mitochondrial biology and the role of canonical IL-6 signaling in muscle cells. Acute exposure of myotubes to IL-6 increased the mitochondrial reactive oxygen species (mtROS) production and oxygen consumption rates (JO2 ) in a manner that was dependent on activation of the JAK/STAT pathway. Furthermore, STAT3 activation by IL-6 was partly attenuated by MitoQ, a mitochondrial-targeted antioxidant, suggesting that mtROS potentiates STAT3 signaling in skeletal muscle in response to IL-6 exposure. In concert with effects on mitochondrial physiology, acute IL-6 exposure induced several mitochondrial adaptations, consistent with the stress-induced mitochondrial hyperfusion. Exposure of myotubes to chronically elevated IL-6 further increased mtROS with eventual loss of respiratory capacity. These data provide new evidence supporting the interplay between cytokine signaling and mitochondrial physiology in skeletal muscle.

TNFα selectively activates the IRE1α/XBP1 endoplasmic reticulum stress pathway in human airway smooth muscle cells.

Airway inflammation is a key aspect of diseases such as asthma. Proinflammatory cytokines such as TNFα mediate the inflammatory response. In various diseases, inflammation leads to endoplasmic reticulum (ER) stress, the accumulation of unfolded proteins, which triggers homeostatic responses to restore normal cellular function. We hypothesized that TNFα triggers ER stress through an increase in reactive oxygen species generation in human airway smooth muscle (hASM) with a downstream effect on mitofusin 2 (Mfn2). In hASM cells isolated from lung specimens incidental to patient surgery, dose- and time-dependent effects of TNFα exposure were assessed. Exposure of hASM to tunicamycin was used as a positive control. Tempol (500 µM) was used as superoxide scavenger. Activation of three ER stress pathways were evaluated by Western blotting: 1) autophosphorylation of inositol-requiring enzyme1 (IRE1α) leading to splicing of X-box binding protein 1 (XBP1); 2) autophosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) leading to phosphorylation of eukaryotic initiation factor 2α; and 3) translocation and cleavage of activating transcription factor 6 (ATF6). We found that exposure of hASM cells to tunicamycin activated all three ER stress pathways. In contrast, TNFα selectively activated the IRE1α/XBP1 pathway in a dose- and time-dependent fashion. Our results indicate that TNFα does not activate the PERK and ATF6 pathways. Exposure of hASM cells to TNFα also decreased Mfn2 protein expression. Concurrent exposure to TNFα and tempol reversed the effect of TNFα on IRE1α phosphorylation and Mfn2 protein expression. Selective activation of the IRE1α/XBP1 pathway in hASM cells after exposure to TNFα may reflect a unique homeostatic role of this pathway in the inflammatory response of hASM cells.

Inflammation-Induced Protein Unfolding in Airway Smooth Muscle Triggers a Homeostatic Response in Mitochondria.

The effects of airway inflammation on airway smooth muscle (ASM) are mediated by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα). In this review article, we will provide a unifying hypothesis for a homeostatic response to airway inflammation that mitigates oxidative stress and thereby provides resilience to ASM. Previous studies have shown that acute exposure to TNFα increases ASM force generation in response to muscarinic stimulation (hyper-reactivity) resulting in increased ATP consumption and increased tension cost. To meet this increased energetic demand, mitochondrial O2 consumption and oxidative phosphorylation increases but at the cost of increased reactive oxygen species (ROS) production (oxidative stress). TNFα-induced oxidative stress results in the accumulation of unfolded proteins in the endoplasmic reticulum (ER) and mitochondria of ASM. In the ER, TNFα selectively phosphorylates inositol-requiring enzyme 1 alpha (pIRE1α) triggering downstream splicing of the transcription factor X-box binding protein 1 (XBP1s); thus, activating the pIRE1α/XBP1s ER stress pathway. Protein unfolding in mitochondria also triggers an unfolded protein response (mtUPR). In our conceptual framework, we hypothesize that activation of these pathways is homeostatically directed towards mitochondrial remodeling via an increase in peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α) expression, which in turn triggers: (1) mitochondrial fragmentation (increased dynamin-related protein-1 (Drp1) and reduced mitofusin-2 (Mfn2) expression) and mitophagy (activation of the Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1)/Parkin mitophagy pathway) to improve mitochondrial quality; (2) reduced Mfn2 also results in a disruption of mitochondrial tethering to the ER and reduced mitochondrial Ca2+ influx; and (3) mitochondrial biogenesis and increased mitochondrial volume density. The homeostatic remodeling of mitochondria results in more efficient O2 consumption and oxidative phosphorylation and reduced ROS formation by individual mitochondrion, while still meeting the increased ATP demand. Thus, the energetic load of hyper-reactivity is shared across the mitochondrial pool within ASM cells.

1α,25-dihydroxyvitamin D3 mitigates cancer cell mediated mitochondrial dysfunction in human skeletal muscle cells.

Cancer cachexia is associated with muscle weakness and atrophy. We investigated whether 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), which has previously been shown to increase skeletal myoblast oxygen consumption rate, could reverse the deleterious effects of tumor cell conditioned medium on myoblast function. Conditioned medium from Lewis lung carcinoma (LLC1) cells inhibits oxygen consumption, increases mitochondrial fragmentation, inhibits pyruvate dehydrogenase activity, and enhances proteasomal activity in human skeletal muscle myoblasts. 1α,25(OH)2D3 reverses the tumor cell-mediated changes in mitochondrial oxygen consumption and proteasomal activity, without changing pyruvate dehydrogenase activity. 1α,25(OH)2D3 might be useful in treatment of weakness seen in association with CC.

TNFα enhances force generation in airway smooth muscle.

Airway inflammation is a hallmark of asthma, triggering airway smooth muscle (ASM) hyperreactivity and airway remodeling. TNFα increases both agonist-induced cytosolic Ca2+ concentration ([Ca2+]cyt) and force in ASM. The effects of TNFα on ASM force may also be due to an increase in Ca2+ sensitivity, cytoskeletal remodeling, and/or changes in contractile protein content. We hypothesized that 24 h of exposure to TNFα increases ASM force by changing actin and myosin heavy chain (MyHC) content and/or polymerization. Porcine ASM strips were permeabilized with 10% Triton X-100, and force was measured in response to increasing concentrations of Ca2+ (pCa 9.0 to 4.0) in control and TNFα-treated groups. Relative phosphorylation of the regulatory myosin light chain (p-MLC) and total actin, MLC, and MyHC concentrations were quantified at pCa 9.0, 6.1, and 4.0. Actin polymerization was quantified by the ratio of filamentous to globular actin at pCa 9.0 and 4.0. For determination of total cross-bridge formation, isometric ATP hydrolysis rate at pCa 4.0 was measured using an enzyme-coupled NADH-linked fluorometric technique. Exposure to TNFα significantly increased force across the range of Ca2+ activation but did not affect the intrinsic Ca2+ sensitivity of force generation. The TNFα-induced increase in ASM force was associated with an increase in total actin, MLC, and MyHC content, as well as an increase in actin polymerization and an increase in maximum isometric ATP hydrolysis rate. The results of this study support our hypothesis that TNFα increases force generation in ASM by increasing the number of contractile units (actin-myosin content) contributing to force generation.

TNFα decreases mitochondrial movement in human airway smooth muscle.

In airway smooth muscle (ASM) cells, excitation-contraction coupling is accomplished via a cascade of events that connect an elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) with cross-bridge attachment and ATP-consuming mechanical work. Excitation-energy coupling is mediated by linkage of the elevation of [Ca2+]cyt to an increase in mitochondrial Ca2+ concentration, which in turn stimulates ATP production. Proximity of mitochondria to the sarcoplasmic reticulum (SR) and plasma membrane is thought to be an important mechanism to facilitate mitochondrial Ca2+ uptake. In this regard, mitochondrial movement in ASM cells may be key in establishing proximity. Mitochondria also move where ATP or Ca2+ buffering is needed. Mitochondrial movement is mediated through interactions with the Miro-Milton molecular complex, which couples mitochondria to kinesin motors at microtubules. We examined mitochondrial movement in human ASM cells and hypothesized that, at basal [Ca2+]cyt levels, mitochondrial movement is necessary to establish proximity of mitochondria to the SR and that, during the transient increase in [Ca2+]cyt induced by agonist stimulation, mitochondrial movement is reduced, thereby promoting transient mitochondrial Ca2+ uptake. We further hypothesized that airway inflammation disrupts basal mitochondrial movement via a reduction in Miro and Milton expression, thereby disrupting the ability of mitochondria to establish proximity to the SR and, thus, reducing transient mitochondrial Ca2+ uptake during agonist activation. The reduced proximity of mitochondria to the SR may affect establishment of transient "hot spots" of higher [Ca2+]cyt at the sites of SR Ca2+ release that are necessary for mitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter.

[Fibromuscular dysplasia and hypertension : beyond renal arteries]. / Hypertension et dysplasie fibromusculaire :au-delà des artères rénales.

Fibromuscular dysplasia (FMD) is a disease associated with abnormalities of the arterial wall of medium-sized arteries. These abnormalities can lead to stenosis or less frequently to dissections or aneurysms. FMD is probably more frequent than initially thought. Nowadays, it is often a chance finding during a radiologic exam. In symptomatic cases, poor organ perfusion due to stenosis, dissection or aneurysm rupture may lead to the diagnosis. The aim of this non-systematic review illustrated with a clinical case is to present our current knowledge of FMD and to highlight the necessity of a standardized and multidisciplinary work-up to improve management of affected patients and understanding of the disease.

La dysplasie fibromusculaire (DFM) est une maladie associée à des anomalies de la paroi des artères de moyen calibre pouvant entraîner des sténoses et plus rarement des anévrismes ou des dissections. Moins rare qu'on ne le pensait initialement, la DFM est parfois découverte fortuitement lors d'un bilan de santé lorsque la maladie est asymptomatique ou lors des manifestations secondaires à l'ischémie de l'organe atteint, conséquence d'une sténose, d'une dissection ou d'une rupture de la paroi artérielle. A partir d'un cas clinique, cette revue non systématique illustre nos connaissances actuelles de la DFM et souligne l'importance d'une prise en charge multidisciplinaire et standardisée pour de meilleurs soins aux patients et une meilleure compréhension de la maladie.

Interaction between endoplasmic/sarcoplasmic reticulum stress (ER/SR stress), mitochondrial signaling and Ca(2+) regulation in airway smooth muscle (ASM).

Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL-13) increase cytosolic Ca(2+) ([Ca(2+)]cyt) responses to agonist stimulation and Ca(2+) sensitivity of force generation, thereby enhancing airway smooth muscle (ASM) contractility (hyper-reactive state). Inflammation also induces ASM proliferation and remodeling (synthetic state). In normal ASM, the transient elevation of [Ca(2+)]cyt induced by agonists leads to a transient increase in mitochondrial Ca(2+) ([Ca(2+)]mito) that may be important in matching ATP production with ATP consumption. In human ASM (hASM) exposed to TNFα and IL-13, the transient increase in [Ca(2+)]mito is blunted despite enhanced [Ca(2+)]cyt responses. We also found that TNFα and IL-13 induce reactive oxidant species (ROS) formation and endoplasmic/sarcoplasmic reticulum (ER/SR) stress (unfolded protein response) in hASM. ER/SR stress in hASM is associated with disruption of mitochondrial coupling with the ER/SR membrane, which relates to reduced mitofusin 2 (Mfn2) expression. Thus, in hASM it appears that TNFα and IL-13 result in ROS formation leading to ER/SR stress, reduced Mfn2 expression, disruption of mitochondrion-ER/SR coupling, decreased mitochondrial Ca(2+) buffering, mitochondrial fragmentation, and increased cell proliferation.

Cigarette smoke-induced mitochondrial fragmentation and dysfunction in human airway smooth muscle.

The balance between mitochondrial fission and fusion is crucial for mitochondria to perform its normal cellular functions. We hypothesized that cigarette smoke (CS) disrupts this balance and enhances mitochondrial dysfunction in the airway. In nonasthmatic human airway smooth muscle (ASM) cells, CS extract (CSE) induced mitochondrial fragmentation and damages their networked morphology in a concentration-dependent fashion, via increased expression of mitochondrial fission protein dynamin-related protein 1 (Drp1) and decreased fusion protein mitofusin (Mfn) 2. CSE effects on Drp1 vs. Mfn2 and mitochondrial network morphology involved reactive oxygen species (ROS), activation of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), protein kinase C (PKC) and proteasome pathways, as well as transcriptional regulation via factors such as NF-κB and nuclear erythroid 2-related factor 2. Inhibiting Drp1 prevented CSE effects on mitochondrial networks and ROS generation, whereas blocking Mfn2 had the opposite, detrimental effect. In ASM from asmatic patients, mitochondria exhibited substantial morphological defects at baseline and showed increased Drp1 but decreased Mfn2 expression, with exacerbating effects of CSE. Overall, these results highlight the importance of mitochondrial networks and their regulation in the context of cellular changes induced by insults such as inflammation (as in asthma) or CS. Altered mitochondrial fission/fusion proteins have a further potential to influence parameters such as ROS and cell proliferation and apoptosis relevant to airway diseases.

Screening and management of hypertensive patients with chronic kidney disease referred to Hypertension Excellence Centres among 27 countries. A pilot survey based on questionnaire.

OBJECTIVE: Real-life management of hypertensive patients with chronic kidney disease (CKD) is unclear. METHODS: A survey was conducted in 2023 by the European Society of Hypertension (ESH) to assess management of CKD patients referred to ESH-Hypertension Excellence Centres (ESH-ECs) at first referral visit. The questionnaire contained 64 questions with which ESH-ECs representatives were asked to estimate preexisting CKD management quality. RESULTS: Overall, 88 ESH-ECs from 27 countries participated (fully completed surveys: 66/88 [75.0%]). ESH-ECs reported that 28% (median, interquartile range: 15-50%) had preexisting CKD, with 10% of them (5-30%) previously referred to a nephrologist, while 30% (15-40%) had resistant hypertension. The reported rate of previous recent (<6 months) estimated glomerular filtration rate (eGFR) and urine albumin-creatinine ratio (UACR) testing were 80% (50-95%) and 30% (15-50%), respectively. The reported use of renin-angiotensin system blockers was 80% (70-90%). When a nephrologist was part of the ESH-EC teams the reported rates SGLT2 inhibitors (27.5% [20-40%] vs. 15% [10-25], P = 0.003), GLP1-RA (10% [10-20%] vs. 5% [5-10%], P = 0.003) and mineralocorticoid receptor antagonists (20% [10-30%] vs. 15% [10-20%], P = 0.05) use were greater as compared to ESH-ECs without nephrologist participation. The rate of reported resistant hypertension, recent eGFR and UACR results and management of CKD patients prior to referral varied widely across countries. CONCLUSIONS: Our estimation indicates deficits regarding CKD screening, use of nephroprotective drugs and referral to nephrologists before referral to ESH-ECs but results varied widely across countries. This information can be used to build specific programs to improve care in hypertensives with CKD.

Effects of the inflammatory cytokines TNF-α and IL-13 on stromal interaction molecule-1 aggregation in human airway smooth muscle intracellular Ca(2+) regulation.

Inflammation elevates intracellular Ca(2+) ([Ca(2+)]i) concentrations in airway smooth muscle (ASM). Store-operated Ca(2+) entry (SOCE) is an important source of [Ca(2+)]i mediated by stromal interaction molecule-1 (STIM1), a sarcoplasmic reticulum (SR) protein. In transducing SR Ca(2+) depletion, STIM1 aggregates to form puncta, thereby activating SOCE via interactions with a Ca(2+) release-activated Ca(2+) channel protein (Orai1) in the plasma membrane. We hypothesized that STIM1 aggregation is enhanced by inflammatory cytokines, thereby augmenting SOCE in human ASM cells. We used real-time fluorescence microscopic imaging to assess the dynamics of STIM1 aggregation and SOCE after exposure to TNF-α or IL-13 in ASM cells overexpressing yellow fluorescent protein-tagged wild-type STIM1 (WT-STIM1) and STIM1 mutants lacking the Ca(2+)-sensing EF-hand (STIM1-D76A), or lacking the cytoplasmic membrane binding site (STIM1ΔK). STIM1 aggregation was analyzed by monitoring puncta size during the SR Ca(2+) depletion induced by cyclopiazonic acid (CPA). We found that puncta size was increased in cells expressing WT-STIM1 after CPA. However, STIM1-D76A constitutively formed puncta, whereas STIM1ΔK failed to form puncta. Furthermore, cytokines increased basal WT-STIM1 puncta size, and the SOCE triggered by SR Ca(2+) depletion was increased in cells expressing WT-STIM1 or STIM1-D76A. Meanwhile, SOCE in cells expressing STIM1ΔK and STIM1 short, interfering RNA (siRNA) was decreased. Similarly, in cells overexpressing STIM1, the siRNA knockdown of Orai1 blunted SOCE. However, exposure to cytokines increased SOCE in all cells, increased basal [Ca(2+)]i, and decreased SR Ca(2+) content. These data suggest that cytokines induce a constitutive increase in STIM1 aggregation that contributes to enhanced SOCE in human ASM after inflammation. Such effects of inflammation on STIM1 aggregations may contribute to airway hyperresponsiveness.

Bioresorbable Magnesium-Based Stent: Real-World Clinical Experience and Feasibility of Follow-Up by Coronary Computed Tomography: A New Window to Look at New Scaffolds.

(1) Background: The diagnostic accuracy of coronary computed tomography angiography (CCTA) for coronary artery disease (CAD) has greatly improved so CCTA represents a transition in the care of patients suffering from CAD. Magnesium-based bioresorbable stents (Mg-BRS) secure acute percutaneous coronary intervention (PCI) results without leaving, in the long term, a metallic caging effect. The purpose of this real-world study was to assess clinical and CCTA medium- and long-term follow-up of all our patients with implanted Mg-BRS. (2) Methods: The patency of 52 Mg-BRS implanted in 44 patients with de novo lesions (24 of which had acute coronary syndrome (ACS)) was evaluated by CCTA and compared to quantitative coronary angiography (QCA) post-implantation. (3) Results: ten events including four deaths occurred during a median follow-up of 48 months. CCTA was interpretable and in-stent measurements were successful at follow-up without being hindered by the stent strut's "blooming effect". Minimal in-stent diameters on CCTA were found to be 1.03 ± 0.60 mm smaller than the expected diameter after post-dilation on implantation (p < 0.05), a difference not found in comparing CCTA and QCA. (4) Conclusions: CCTA follow-up of implanted Mg-BRS is fully interpretable and we confirm the long-term Mg-BRS safety profile.