Function

Skeletal muscles and blood vessels are continuously exposed to mechanical forces, particularly during exercise. We subjected human endothelial and skeletal muscle cells to cyclic mechanical stretch and investigated acute molecular responses. Mechanical loading elicited both shared and cell type-specific alterations in transcriptomic and metabolomic profiles, several of which mirror changes observed in vivo following exercise. Notably, both cell types released acetate in response to mechanical loading, however, many of the changes occurred in opposite directions. For example, genes associated with the electron transport chain were repressed in endothelial cells but upregulated in skeletal muscle cells. Furthermore, mechanical loading promoted a transcriptomic shift in endothelial cells indicative of maturation, and reduced their proliferation. Metabolic changes were more pronounced in endothelial cells, which exhibited increased serine biosynthesis from glucose, as demonstrated by 13C-(U)-glucose tracing. Further experiments targeting phosphoglycerate dehydrogenase (PHGDH), a key enzyme in the serine synthesis pathway, underscored its role in endothelial cell anabolism. These findings suggest that mechanical loading alone can recapitulate several exercise-induced effects in endothelial and muscle cells, and highlight a potential link between mechanical stimuli, serine synthesis, and endothelial cell maturation.

Elephant seals experience extreme hypoxemia during diving bouts. Similar depletions in oxygen availability characterize pathologies including myocardial infarction and ischemic stroke in humans, but seals manage these repeated episodes without injury. However, the real-time assessment of the molecular changes underlying protection against hypoxic injury in seals remains restricted by their at-sea inaccessibility. Hence, we developed a proliferative arterial endothelial cell culture system to assess the molecular response to prolonged hypoxia. Seal and human cells exposed to 1% O2 for up to 6 h demonstrated differential responses to both acute and prolonged hypoxia. Seal cells decouple stabilization of the hypoxia-sensitive transcriptional regulator HIF-1 from angiogenic signaling at both the transcriptional and cellular level. Rapid upregulation of genes involved in the glutathione (GSH) metabolism pathway supported maintenance of GSH pools and increases in intracellular succinate in seal but not human cells during hypoxia exposure. High maximal and spare respiratory capacity in seal cells after hypoxia exposure occurred in concert with increasing mitochondrial branch length and independent from major changes in extracellular acidification rate, suggesting seal cells recover oxidative metabolism without significant glycolytic dependency after hypoxia exposure. In sum, our studies show that in contrast to human cells, seal cells adapt to hypoxia exposure by dampening angiogenic signaling, increasing antioxidant protection, and maintaining mitochondrial morphological integrity and function.

BackgroundIn vascular smooth muscle cells (VSMCs), LRRC8A volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis factor alpha (TNF), angiotensin II and stretch. LRRC8A physically associates with NADPH oxidase 1 (Nox1) and supports its production of extracellular superoxide (O2-*). Methods and ResultsMice lacking LRRC8A exclusively in VSMCs (Sm22-Cre, KO) were used to assess the role of VRACs in TNF signaling and vasomotor function. KO mesenteric vessels contracted normally to KCl and phenylephrine, but relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) was enhanced compared to wild type (WT). 48 hours of ex vivo exposure to TNF (10ng/ml) markedly impaired dilation to ACh and SNP in WT but not KO vessels. VRAC blockade (carbenoxolone, CBX, 100 M, 20 min) enhanced dilation of control rings and restored impaired dilation following TNF exposure. Myogenic tone was absent in KO rings. LRRC8A immunoprecipitation followed by mass spectroscopy identified 35 proteins that interacted with LRRC8A. Pathway analysis revealed actin cytoskeletal regulation as the most closely associated function of these proteins. Among these proteins, the Myosin Phosphatase Rho-Interacting protein (MPRIP) links RhoA, MYPT1 and actin. LRRC8A-MPRIP co-localization was confirmed by confocal imaging of tagged proteins, Proximity Ligation Assays, and IP/western blots which revealed LRRC8A binding at the second Pleckstrin Homology domain of MPRIP. siLRRC8A or CBX treatment decreased RhoA activity in cultured VSMCs, and MYPT1 phosphorylation at T853 was reduced in KO mesenteries suggesting that reduced ROCK activity contributes to enhanced relaxation. MPRIP was a target of redox modification, becoming oxidized (sulfenylated) after TNF exposure. ConclusionsInteraction of Nox1/LRRC8A with MPRIP/RhoA/MYPT1/actin may allow redox regulation of the cytoskeleton and link Nox1 activation to both inflammation and vascular contractility.

Sodium (Na+) and potassium (K+) movements during repetitive stimulation of skeletal muscle fibers leads to lowered transmembrane Na+ and K+ gradients. Impaired calcium release resulting from the predicted reduction of the action potential (AP) overshoot (OS) has been suggested as a causative factor of muscle fatigue. To test this hypothesis, we used a double grease-gap method and simultaneously recorded membrane action potentials (MAPs) and Ca2+ release (as Ca2+ transients), elicited by single pulses or short trains of pulses (100 Hz, 100 ms), in rested fibers polarized to membrane potentials (Vm) between -100 to -55 mV, and exposed to various extracellular Na+ concentrations ([Na+]o; 115, 90, 60 and 40 mM). In single stimulation experiments, we found that at physiological Vm (-100 mV), Ca2+ release was mostly immune to [Na+]o reductions up to 60 mM (~1/2 the physiological value). In contrast, at 40 mM Na+o Ca2+ release was reduced by 80%, notwithstanding robust MAPs with large OS (~30 mV) were recruited in this conditions. At Vm between -100 and -60 mV, a 20% reduction of [Na+]o (115 to 90 mM) had no major detrimental effects on Ca2+ release. Instead, depolarization-dependent potentiation of Ca2+ transients, with a maximum at -65 mV, was observed at both 115 and 90 mM Na+o. Potentiation was smaller at 90 mM Na+o. At both [Na+]o, maximally potentiated Ca2+ transients (i.e. at -60 mV) were recruited by MAPS with reduced OSs. In contrast, Ca2+ release was significantly depressed and no potentiation was observed at Vm between -100 to -70 mV when [Na+]o was reduced 60 mM. At extreme Na+o (40 mM), Ca2+ release recorded at Vm between -100 and -70 mV was almost obliterated; nonetheless robust MAPs, with OSs of ~25 mV, were recruited. Extreme depolarizations significantly depressed Ca2+ release at all [Na+]o tested. The Vm leading to Ca2+ release depression was more negative the lower the [Na+]o (-55, -60 and -70 for 115, 90 and mM Na+o, respectively). Fiber exposed to 115-60 mM Na+o can sustain normal Ca2+ release at a frequency of 100 Hz when polarized between -100 and -80 mV. Depolarizations beyond -80 mV lead to impaired Ca2+ release along the trains. In most cases, there was no correlation between changes in Ca2+ release and changes in OS. At 40 mM Na+o, only the 1st-3rd stimuli of trains recruited Ca2+ transients, which were significantly depressed vis a vis close to normal MAPs. Neither the OS nor the duration of MAPs are figures of merit predicting the amplitude of Ca2+ transients. At critical combinations of depolarization, [Na+]o, and stimulation frequency, potentiated Ca2+ transients are recruited by MAPS with small OSs; and conversely, partial or total decoupling of Ca2+ release from close to normal MAPs was observed. Depolarization and Na+o deprivation depressed Ca2+ release in a synergistic way; lowered [Na+]o increased the detrimental effects of depolarization on Ca2+ release, and depolarization render the ECC process more sensitivity to Na+o deprivation. Impaired TTS AP generation and/or conduction may explain the detrimental effects of depolarization and Na+o deprivation on Ca2+ release. The effects of increased K+o and Na+o deprivation on the force generation of rested fibers can be explained on the basis of the effects of membrane depolarization and Na+o deprivation on Ca2+ release. Definitions[ion]i, [ion]o: intracellular and extracellular ion concentrations; ion= Na+, K+, Ca+2. (in molar units) EFM-Na, EMF-K: electromotive force of Na+ and K+ (in mV) ENa, EK: equilibrium potential for Na+ and Na+ (in mV) Vm: membrane or holding potential (in mV) TTS: transverse tubular system. Ca-FWHM, Ca+2 transient full-width at half-maximum (in ms) MAP-FWHM: MAP full-width at half-maximum (in ms) REF: releasing effective time, time a MAP waveform is above -40 mV (in ms)

Respiration and blood pressure (BP) are regulated to maintain optimal delivery of O2 to every cell in the body. Arterial hypoxemia is sensed by the carotid body (CB), which initiates sympathetic reflex arcs to the diaphragm to increase ventilation, and to the adrenal medulla (AM) to increase catecholamine secretion and thereby increase BP. However, the underlying molecular mechanisms have not been fully delineated. Here, we report that the relative activities of hypoxia-inducible factor-1 (HIF-1) and HIF-2 determine the set point for the CB and AM, with respect to their maintenance of BP and respiration. In Hif2a+/- mice, which are heterozygous for a knockout allele at the locus encoding HIF-2, expression of HIF-1 and NADPH oxidase 2 was increased in the CB and AM, resulting in an oxidized intracellular redox state with augmented sensitivity to hypoxia, increased BP, and respiratory abnormalities, which were all normalized by treatment with a HIF-1 inhibitor or a superoxide anion scavenger. By contrast, in Hif1a+/- mice, which are heterozygous for a knockout allele at the locus encoding HIF-1, the expression of HIF-2 and superoxide dismutase 2 was increased in the CB and AM, resulting in a reduced intracellular redox state with impaired CB and ventilatory responses to chronic hypoxia, which were normalized by treatment with a HIF-2 inhibitor. None of the abnormalities that were observed in Hif1a+/- or Hif2a+/- mice were observed in Hif1a+/-; Hif2a+/- double- heterozygous mice. Our results demonstrate that redox balance in the CB and AM, which is determined by mutual antagonism between HIF- isoforms, establishes the set point for responses of the CB and AM to hypoxia, and is required for the maintenance of normal BP and respiration.

Many have believed that oxygen (O2) crosses red blood cell (RBC) membranes by dissolving in lipids that offer no resistance to diffusion. However, using stopped-flow (SF) analyses of hemoglobin (Hb) absorbance spectra during O2 off-loading from mouse RBCs, we now report that most O2 traverses membrane-protein channels. Two agents excluded from the RBC interior markedly slow O2 off-loading: p-chloromercuribenzenesulfonate (pCMBS) reduces inferred membrane O2 permeability (PMembrane) by [~]82%, and 4,4-diisothiocyanatostilbene-2,2-disulfonate (DIDS), by [~]56%. Because neither likely produces these effects via membrane lipids, we examined RBCs from mice genetically deficient in aquaporin-1 (AQP1), the Rh complex (i.e., rhesus proteins RhAG + mRh), or both. The double knockout (dKO) reduces PMembrane by [~]55%, and pCMBS+dKO, by [~]91%. Proteomic analyses of RBC membranes, flow cytometry, hematology, and mathematical simulations rule out explanations involving other membrane proteins, RBC geometry, or extracellular unconvected fluid (EUF). By identifying the first two O2 channels and pointing to the existence of other O2 channel(s), all of which could be subject to physiological regulation and pharmacological intervention, our work represents a paradigm shift for O2 handling.

Under healthy conditions the urinary bladder undergoes relatively long periods of filling with well-spaced voiding events to ensure proper storage and removal of urine respectively. During the filling phase, distinct contractile events in detrusor urinary smooth muscle (UBSM) elicit transient non-voiding pressure events and associated bursts in afferent nerve activity to relay the sensation of bladder fullness. The mechanisms that regulate UBSM excitability and associated non-voiding pressure events under physiological and pathological conditions are poorly understood. Here we investigated the role of adenosine signaling in regulating urinary bladder contractility. Using an ex vivo pressurized bladder preparation from mice and patch-clamp electrophysiology in isolated UBSM we evaluated whole bladder transient pressure events, whole bladder detrusor Ca2+ activity, and single UBSM ion channel activity. We found that adenosine suppresses bladder activity through activation of A2B adenosine receptors and downstream activation of large-conductance calcium-activated potassium (BKCa) channels. We further demonstrated that acute exposure to low oxygen conditions using a chemical oxygen scavenger potently suppresses bladder contractility through the A2B receptor pathway. These results highlight the prominent role adenosine receptors and downstream potassium channels play in regulating urinary bladder contractility in physiological and pathological contexts.

Increased red cell distribution width (RDW), which measures erythrocyte size variability (anisocytosis), has been linked to early mortality in many diseases and normal aged population through unknown mechanisms. Hypoxia has been proposed to increase both RDW and mortality. However, experimental evidence, especially in animal models, is lacking. Here, we show that chronic hypobaric hypoxia (~10% O2) increases erythrocyte numbers, hemoglobin and RDW, while reducing longevity in male mice. Compound heterozygous knockout (chKO) mutations in succinate dehydrogenase (Sdh; mitochondrial complex II) genes Sdhb, Sdhc and Sdhd reduce high RDW and immature reticulocyte fraction, and increase healthy lifespan in chronic hypoxia. Hemoglobin and erythrocyte numbers in hypoxia do not show statistically significant differences between Sdh chKO and WT mice. These results identify a mitochondrial mechanism regulating both RDW and organismal adaptation to chronic hypoxia, and suggest SDH as a potential therapeutic target to reduce high RDW-associated clinical mortality.

Exercise modulates vascular plasticity in multiple organ systems; however, the metabolomic transducers underlying exercise and vascular protection in the disturbed flow-prone vasculature remain under-investigated. We simulated exercise-augmented pulsatile shear stress (PSS) to mitigate flow recirculation in the lesser curvature of the aortic arch. When human aortic endothelial cells (HAECs) were subjected to PSS ({tau}ave = 50 dyne{middle dot}cm-2, {partial}{tau}/{partial}t = 71 dyne{middle dot}cm-2{middle dot}s-1, 1 Hz), untargeted metabolomic analysis revealed that Stearoyl-CoA Desaturase (SCD1) in the endoplasmic reticulum (ER) catalyzed the fatty acid metabolite, oleic acid (OA), to mitigate inflammatory mediators. Following 24 hours of exercise, wild-type C57BL/6J mice developed elevated SCD1-catalyzed lipid metabolites in the plasma, including OA and palmitoleic acid (PA). Exercise over a 2-week period increased endothelial SCD1 in the ER. Exercise further modulated the time-averaged wall shear stress (TAWSS or{tau} ave) and oscillatory shear index (OSIave), upregulated Scd1 and attenuated VCAM1 expression in the disturbed flow-prone aortic arch in Ldlr-/- mice on high-fat diet but not in Ldlr-/-Scd1EC-/- mice. Scd1 overexpression via recombinant adenovirus also mitigated ER stress. Single cell transcriptomic analysis of the mouse aorta revealed interconnection of Scd1 with mechanosensitive genes, namely Irs2, Acox1 and Adipor2 that modulate lipid metabolism pathways. Taken together, exercise modulates PSS ({tau}ave and OSIave) to activate SCD1 as a metabolomic transducer to ameliorate inflammation in the disturbed flow-prone vasculature.

VASP is a member of the Enabled/VASP protein family that is involved in cortical actin dynamics and may also contribute to the formation of gap junctions. In vessels, gap junctional coupling allows the transfer of signals along the vessel wall and coordinates vascular behavior. Moreover, VASP is reportedly a mediator of NO-induced inhibition of platelet aggregation. Therefore, we hypothesized that VASP exerts also important physiologic functions in arterioles. We examined the spread of vasodilations enabled by gap junctional coupling in endothelial cells as well as NO-induced arteriolar dilations in VASP-deficient mice by intravital microscopy of the microcirculation in a skeletal muscle in anesthetized mice. Conducted dilations were initiated by brief, locally confined stimulation of the arterioles with acetylcholine. The maximal diameters of the arterioles under study ranged from 30 to 40 m. Brief stimulation with acetylcholine induced a short dilation at the local site that was also observed at remote, upstream sites without an attenuation of the amplitude up to a distance of 1.2 mm in control animals (wild-type). In contrast, remote dilations were reduced in VASP-deficient mice despite a similar local dilation indicating an impairment of conducted dilations. Superfusion of NOdonors induced a concentration-dependent dilation in wild-type mice. However, these dilations were slightly reduced in VASP-deficient animals. In contrast, dilations induced by the endothelial stimulator acetylcholine were fully preserved in VASP-deficient mice. In summary, this study suggests that VASP exerts critical functions in arteriolar diameter control. It is crucial for the conduction of dilator signals along the endothelial cell layer. The impairment possibly reflects a perturbed formation of gap junctions in the endothelial cell membrane. VASP also participates in the full dilatory potential of NOdonors although the effect of its deficiency is only subtle. In contrast, VASP is not required for dilations initiated by endothelial stimulation which are mediated in the murine microcirculation by an EDH-mechanism.

Recent studies have emphasized the importance of the nitric oxide synthase (NOS)-independent, nitrate (NO3-) [->] nitrite (NO2-) [->] nitric oxide (NO) pathway in skeletal muscle. In particular, it has been hypothesized that this pathway is especially active in type II, or fast-twitch, muscle fibers, necessitating greater NO3- and NO2- storage. We therefore measured NO3- and NO2- concentrations in the predominantly fast-twitch vastus lateralis and predominantly slow-twitch soleus muscles of rats. Contrary to the above hypothesis, we found that NO3- and NO2- concentrations were 3.4-fold and 1.8-fold higher, respectively, in the soleus. On the other hand, NO signaling (i.e., cyclic guanosine monophosphate (cGMP) level) was comparable in the two muscles. Although the physiological significance of these observations remains to be determined, we speculate that NO production via the NO3- [->] NO2- [->] NO pathway is normally higher in slow-twitch muscles, thus helping compensate for their inherently lower NOS activity.

Nicotinamide adenine dinucleotide (NAD+) plays a central role in energy metabolism, and its decline is linked to various degenerative diseases. While NAD+ restoration holds therapeutic promise, its long term, tissue-specific consequences remain poorly understood. We investigated effects of nicotinamide riboside (NR) supplementation for "mutator" mice manifesting mitochondrial progeria. Our results reveal strikingly divergent outcomes: in proliferative bone marrow, NR-treated mutators show reductive stress with accumulation of NADH/NADPH, altered amino acid, nucleotide, folate levels and impaired heme biosynthesis. In blood, erythrocyte maturation defects are aggravated, exacerbating anemia. Conversely, in postmitotic cardiac tissue, NR enhanced contractility, reduces stress response markers and normalized metabolic profile. These findings indicate that while being beneficial for heart, chronic NAD+ boosting can compromise erythrocyte maturation in the context of mitochondrial disease. The data emphasize importance of evaluating systemic effects of NAD+ boosting therapies beyond the primary affected tissues and development of tissue-specific metabolic interventions for degenerative diseases. HighlightsO_LIChronic nicotinamide riboside supplementation exacerbates anemia and disrupts erythroid maturation in progeric mice. C_LIO_LIIn proliferative bone marrow cells, NR induces redox imbalance and drives profound metabolic dysregulation. C_LIO_LINR suppresses heme biosynthesis and iron transport pathways in the bone marrow. C_LIO_LIIn the heart, NR restores NAD+ levels, enhances cardiac function, and reduces metabolic stress. C_LI

S-nitrosylation of Cx43 gap junction channels critically regulates communication between smooth muscle cells and endothelial cells. This posttranslational modification also induces the opening of undocked Cx43 hemichannels. However, its specific impact on vasomotor regulation remains unclear. Considering the role of endothelial TRPV4 channel activation in promoting vasodilation through nitric oxide (NO) production, we investigated the direct modulation of endothelial Cx43 hemichannels by TRPV4 channel activation. Using the proximity ligation assay, we identify that Cx43 and TRPV4 are found in close proximity in the endothelium of resistance arteries. In primary endothelial cell cultures from resistance arteries (ECs), GSK-induced TRPV4 activation enhances eNOS activity, increases NO production, and opens Cx43 hemichannels via direct S-nitrosylation. Notably, the elevated intracellular Ca2+ levels caused by TRPV4 activation were reduced by blocking Cx43 hemichannels. In ex vivo mesenteric arteries, inhibiting Cx43 hemichannels reduced endothelial hyperpolarization without affecting NO production in ECs, underscoring a critical role of TRPV4/Cx43 signaling in endothelial electrical behavior. We perturbed the proximity of Cx43/TRPV4 by disrupting lipid rafts in ECs using {beta}-cyclodextrin. Under these conditions, hemichannel activity, Ca2+ influx, and endothelial hyperpolarization were blunted upon GSK stimulation. Intravital microscopy of mesenteric arterioles in vivo further demonstrated that inhibiting Cx43 hemichannels activity, NO production and disrupting endothelial integrity reduce TRPV4-induced relaxation. These findings underscore a new pivotal role of Cx43 hemichannel associated with TRPV4 signaling pathway in modulating endothelial electrical behavior and vasomotor tone regulation.

BACKGROUNDVascular endothelial cells (ECs) experience two different blood flow patterns: laminar and disturbed flows. Their responses to laminar flow contribute to vascular homeostasis, whereas their responses to disturbed flow result in EC dysfunction and vascular diseases. However, it remains unclear how ECs differentially sense laminar and disturbed flows and trigger signalings that elicit different EC responses. We aimed to investigate EC flow-sensing and signaling mechanisms, focusing on the role of the plasma membrane and mitochondria. METHODSWe exposed cultured human aortic ECs to laminar flow and disturbed flow in flow-loading devices and used real-time imaging with optical probes to examine changes in the lipid order of the plasma and mitochondria membranes and the mitochondrial adenosine triphosphate (ATP) production and hydrogen peroxide (H2O2) release. RESULTSThe lipid order of EC plasma membranes immediately decreased in response to laminar flow, while it increased in response to disturbed flow. Laminar flow also decreased the lipid order of mitochondrial membranes and increased mitochondrial ATP production. In contrast, disturbed flow increased the lipid order of mitochondrial membranes and increased the release of H2O2 from mitochondria. Addition of cholesterol to the cells increased the lipid order of both membranes and abrogated the laminar flow-induced ATP production, while treatment of the cells with a cholesterol-depleting reagent, methyl-{beta} cyclodextrin, decreased the lipid order of both membranes and abolished the disturbed flow-induced H2O2 release, indicating that the changes in the membrane lipid order are closely linked to the flow-induced changes in the mitochondrial functions. CONCLUSIONSECs differentially sense laminar and disturbed flows by altering the lipid order of their plasma and mitochondrial membranes in opposite directions, which result in distinct changes in the mitochondrial functions, namely, increased ATP production for laminar flow and increased H2O2 release for disturbed flow, leading to ATP- and H2O2-mediated signalings, respectively.

The vascular smooth muscle (VSM) of resistance blood vessels displays intrinsic autoregulatory responses to increased intraluminal pressure, the myogenic response. In the brain, the myogenic responses of cerebral arterioles are critical to homeostatic blood flow regulation. Here we provide the first evidence to link the death-associated protein kinase 3 (DAPK3) to the myogenic response of rat and human cerebral arterioles. DAPK3 is a Ser/Thr kinase involved in Ca2+- sensitization mechanisms of VSM contraction. Ex vivo administration of a specific DAPK3 inhibitor (i.e., HS38) could attenuate vessel constrictions invoked by serotonin as well as intraluminal pressure elevation. The HS38-dependent dilation was not associated with any change in myosin light chain (LC20) phosphorylation. The results suggest that DAPK3 does not regulate Ca2+ sensitization pathways during the myogenic response of cerebral vessels but rather operates to control the actin cytoskeleton. Finally, a slow return of myogenic tone was observed during the sustained exposure of cerebral arterioles to a suite of DAPK3 inhibitors. Recovery of tone was associated with greater LC20 phosphorylation that suggests intrinsic signaling compensation in response to attenuation of DAPK3 activity. The translational importance of DAPK3 to the human cerebral vasculature was noted, with robust expression of the protein kinase and significant HS38-dependent attenuation of myogenic reactivity found for human pial vessels.

IKK2-NF{kappa}B pathway mediated-inflammation in vascular smooth muscle cells (VSMCs) has been proposed to be an etiologic factor in medial calcification and stiffness. However, the role of the IKK2-NF{kappa}B pathway in medial calcification remains to be elucidated. In this study, we found that CKD induces inflammatory pathways through the local activation of the IKK2-NF{kappa}B pathway in VMSCs associated with calcified vascular stiffness. Despite reducing the expression of inflammatory mediators, complete inhibition of the IKK2-NF{kappa}B pathway in vitro and in vivo unexpectedly exacerbated vascular mineralization and stiffness. In contrast, activation of NF{kappa}B by SMC-specific I{kappa}B deficiency attenuated calcified vascular stiffness in CKD. Inhibition of the IKK2-NF{kappa}B pathway induced apoptosis of VSMCs by reducing anti-apoptotic gene expression, whereas activation of NF{kappa}B reduced CKD-dependent vascular cell death. In addition, increased calcifying extracellular vesicles through the inhibition of the IKK2-NF{kappa}B pathway induced mineralization of VSMCs, which was significantly reduced by blocking cell death. This study reveals that activation of the IKK2-NF{kappa}B pathway in VSMCs plays a protective role in CKD-dependent calcified vascular stiffness by reducing the release of apoptotic calcifying extracellular vesicles.

Enabled/vasodilator stimulated phosphoproteins (Ena/VASP) proteins are important regulators of the cytoskeleton, linking kinase signaling pathways to actin assembly. In mammals, the Ena/VASP family of proteins consists of mammalian enabled (Mena), VASP, and Ena-VASP-like protein (EVL). The proteins are well known targets of cAMP- and cGMP-dependent protein kinases, PKA and PKG, respectively. Given the importance of cyclic nucleotide signaling in mediating vasodilation, we investigated the role of Ena/VASP protein in vascular smooth muscle relaxation. Whereas VASP and Mena were strongly expressed in vascular smooth muscle cells, EVL was undetectable in the arterial wall and EVL-deficiency had no impact on agonist-induced smooth muscle relaxation. VASP deletion impaired the acetylcholine (ACh)- and nitric oxide (NO)-induced relaxation murine mesenteric arteries ex vivo. Similarly, the ACh-induced and NO-dependent relaxation of aorta from 7-month-old but not 3- month-old VASP-/- mice was also reduced. Aortas from animals lacking VASP and expressing only minimal amounts of Mena displayed significantly impaired relaxations in response to NO, cAMP and cGMP stimulation. These results suggest that Mena and VASP play an important role in agonist induced smooth muscle relaxation and functionally compensate for each other.

Mechanisms by which disuse promotes skeletal muscle atrophy is not well understood. We previously demonstrated that disuse reduces the abundance of mitochondrial phosphatidylethanolamine (PE) in skeletal muscle. Deletion of phosphatidylserine decarboxylase (PSD), an enzyme that generates mitochondrial PE, was sufficient to promote muscle atrophy. In this study, we tested the hypothesis that muscle atrophy induced by PSD deletion is driven by an accumulation of lipid hydroperoxides (LOOH). Mice with muscle-specific knockout of PSD (PSD-MKO) were crossed with glutathione peroxidase 4 (GPx4) transgenic mice (GPx4Tg) to suppress the accumulation of LOOH. However, PSD-MKO x GPx4Tg mice and PSD-MKO mice demonstrated equally robust loss of muscle mass. These results suggest that muscle atrophy induced by PSD deficiency is not driven by the accumulation of LOOH.

BackgroundIntimal hyperplasia (IH) remains a major limitation in the long-term success of any type of revascularization. IH is due to vascular smooth muscle cell (VSMC) dedifferentiation, proliferation and migration. The gasotransmitter Hydrogen Sulfide (H2S) inhibits IH in pre-clinical models. However, there is currently no clinically approved H2S donor. Here we used sodium thiosulfate (STS), a clinically-approved source of sulfur, to limit IH. MethodsHypercholesterolemic LDLR deleted (LDLR-/-), WT or CSE-/- male mice randomly treated with 4g/L STS in the water bottle were submitted to focal carotid artery stenosis to induce IH. Human vein segments were maintained in culture for 7 days to induce IH. Further in vitro studies were conducted in primary human vascular smooth muscle cell (VSMC). FindingsSTS inhibited IH in mice and in human vein segments. STS inhibited cell proliferation in the carotid artery wall and in human vein segments. STS increased polysulfides in vivo and protein persulfidation in vitro, which correlated with microtubule depolymerization, cell cycle arrest and reduced VSMC migration and proliferation. InterpretationSTS, a drug used for the treatment of cyanide poisoning and calciphylaxis, protects against IH in a mouse model of arterial restenosis and in human vein segments. STS acts as an H2S donor to limit VSMC migration and proliferation via microtubule depolymerization. FundingThis work was supported by the Swiss National Science Foundation (grant FN-310030_176158 to FA and SD and PZ00P3-185927 to AL); the Novartis Foundation to FA; and the Union des Societes Suisses des Maladies Vasculaires to SD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/459573v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@c98479org.highwire.dtl.DTLVardef@1863f0org.highwire.dtl.DTLVardef@1492ad7org.highwire.dtl.DTLVardef@9bca8a_HPS_FORMAT_FIGEXP M_FIG C_FIG Research in contextO_ST_ABSEvidence before this studyC_ST_ABSIntimal hyperplasia (IH) is a complex process leading to vessel restenosis, a major complication following cardiovascular surgeries and angioplasties. Therapies to limit IH are currently limited. Pre-clinical studies suggest that hydrogen sulfide (H2S), an endogenous gasotransmitter, limits restenosis. However, despite these potent cardiovascular benefits in pre-clinical studies, H2S-based therapeutics are not available yet. Sodium thiosulfate (Na2S2O3) is an FDA-approved drug used for the treatment of cyanide poisoning and calciphylaxis, a rare condition of vascular calcification affecting patients with end-stage renal disease. Evidence suggest that thiosulfate may generate H2S in vivo in pre-clinical studies. Added value of this studyHere, we demonstrate that STS inhibit IH in a surgical mouse model of IH and in an ex vivo model of IH in human vein culture. We further found that STS increases circulating polysulfide levels in vivo and inhibits IH via decreased cell proliferation via disruption of the normal cells cytoskeleton. Finally, using CSE knockout mice, the main enzyme responsible for H2S production in the vasculature, we found that STS rescue these mice from accelerated IF formation. Implications of all the available evidenceThese findings suggest that STS holds strong translational potentials to limit IH following vascular surgeries and should be investigated further.

Acute kidney injury (AKI) is a prevalent and significant complication in critically ill patients, and its management remains a considerable challenge. The kidney is a highly metabolic organ, consuming and producing substantial amounts of ATP, mainly through mitochondrial oxidative phosphorylation. Recently, mitochondrial dysfunction has been identified as a key factor in the pathophysiology of AKI and the progression to chronic kidney disease. The kidney is a complex organ, comprising millions of structural and functional units. These nephrons are composed of different cell types dwelling within specific metabolic microenvironment. Whether the metabolic spatialization in the kidney has consequences on tubular injury distribution and severity remains unclear. In this study, we identified the high metabolic rate of the outer stripe of the outer medulla (OSOM) and its substrate preference flexibility, relying on both glycolysis and fatty acid oxidation (FAO) to fulfill its ATP demands. We demonstrated that the OSOM is susceptible to mitochondrial and FAO impairment induced by propofol, the most used sedative in intensive care settings, which exacerbates tubular injury during AKI. In the clinical setting, the cumulative dose of propofol is positively correlated with oxidative metabolism disruption and histological and function outcomes in renal allograft recipients. Finally, we found that the loop of Henle, the OSOM major constituent, was the most injured segment during AKI in patients. This study shows how renal metabolic spatialization impacts tubular injury severity. We identified the OSOM as the most metabolically active and the most injured region of the kidney both in humans and mice. We demonstrated that propofol is a potent inhibitor of renal mitochondrial function and FAO exacerbating tubular injury in the OSOM upon IRI. Translational StatementO_LIAerobic metabolism is basally enhanced in the renal OSOM, including the S3 proximal tubule, the thick ascending limb of the loop of Henle and Distal Convoluted Tubule C_LIO_LIPT cells as well as TAL cells are significantly targeted by injury in human AKI. C_LIO_LIPropofol impairs renal mitochondrial function worsening tubular injury during ischemia reperfusion. C_LI