Function

Cerebral blood flow (CBF) control is essential for normal brain function and is disrupted in pathological conditions. Arterial diameters are tightly regulated to provide on demand increases in blood flow in regions of neuronal activity. Pericytes (PCs) exhibit robust myogenic tone and may also respond to neuronal activity to fine-tune local resistance and blood flow. Thus, mural control of microcirculatory resistance may extend beyond arteries and arterioles. Yet, PCs electrophysiology and contractility have not been thoroughly characterized, and this prohibits an integrated view of brain blood flow control. In this study, we develop a detailed mathematical model of mural cell electrophysiology, Ca2+ dynamics and biomechanics. The model is informed by electrophysiological data in smooth muscle cells (SMCs) or PCs and predictions are compared against pressure-induced responses in isolated arterioles and capillaries, respectively. Simulations recapitulate myogenic constrictions and examine differences in contractile dynamics as we move from arterioles to proximal and distal capillaries. In arteriole-to-capillary transitional (ACT) zone PCs, increased mechanosensitivity, more Ca2+ influx through non-selective cation (NSC) channels and/or a higher sensitivity of the contractile apparatus to Ca2+ can compensate for reduced L-type voltage-operated (VOCC) Ca2+ influx and allow for robust constrictions at the lower operating pressures of capillaries relative to the arterioles. A significant Ca2+ influx through NSC relative to VOCC, however, can decouple the PCs contractile apparatus from electrical signaling. Vasoactivity to chemomechanical stimuli along the arteriole to capillary axis is progressively driven by VOCC-independent Ca2+ influx and Ca2+ sensitization with slow kinetics. The proposed cell model can form the basis for detailed multiscale and multicellular models that will examine physiological function at a single vessel or vascular network levels and investigate CBF control in health and in disease. Key pointsO_LIA mural cell model of electrophysiology, calcium (Ca2+) dynamics and biomechanics is informed by data and adapted for modeling cerebral arteriole smooth muscle cells and capillary pericytes. C_LIO_LIIon channel activities are characterized by patch-clamp electrophysiology in isolated cerebral smooth muscle cell and pericytes, and capillary and arteriole electromechanical responses to transmural pressure changes are assessed using novel ex vivo preparations. C_LIO_LIMyogenic constrictions in arterioles can be reproduced by pressure-induced non-selective cation channel (NSC) activation that depolarizes the cell, opens L-type Ca2+ channels (VOCCs) and increases Ca2+ influx. C_LIO_LIRobust myogenic constrictions in arteriole-to-capillary transition (ACT) zone pericytes may reflect significant Ca2+ influx through NSC, increased mechanosensitivity, or higher sensitivity of the contractile apparatus to Ca2+, potentially compensating for reduced VOCC density relative to arteriolar smooth muscle. C_LIO_LIA significant contribution of NSC relative to VOCC in Ca2+ influx, can decouple the contractile apparatus from electrical signaling. C_LIO_LIThe model shows how gradients in ionic activities, mechanosensitivity and/or Ca2+ sensitivity can alter contractile phenotype and electromechanical coupling along the arteriole to capillary continuum. C_LIO_LIThe proposed model can form the basis for detailed multiscale and multicellular models that will investigate cerebral blood flow control in health and in disease. C_LI

BackgroundCoronary autoregulation is the ability of the normal heart to maintain constant coronary blood flow (CBF) over a wide range of coronary driving pressures (CDP). Despite being vital for survival, the mechanism of coronary autoregulation is unknown. We hypothesized that GPR39, present in vascular smooth muscle cells, together with its endogenous agonist 15-hydroxyeicosatetraenoic acid (15-HETE) orchestrate coronary autoregulation. MethodsWe created coronary stenoses of varying degrees in open-chest, anesthetized dogs where we measured CBF and CDP. In a subset of animals, coronary venous blood was sampled for eicosanoid, adenosine, endothelin-1, polyunsaturated fatty acids, and prostaglandins levels. Stenoses were recreated during intravenous administration of VC108, a specific GPR39 antagonist and systemic, pulmonary, and coronary hemodynamics measured. ResultsGPR39 was identified in coronary arterioles by immunohistochemistry and in heart tissue by western blot. In-vivo, 15-HETE correlated linearly with CDP over the autoregulatory range (r2=0.47, p=0.0024). Apart from 6-keto PGF1 no other metabolite had any relation with CDP. Prior to administration of VC108, CBF did not change within the autoregulatory range. VC108 had no effect of systemic and pulmonary hemodynamics but increased CBF (p=0.02 versus vehicle) by decreasing coronary microvascular resistance (p=0.01 versus vehicle), indicating that GPR39 participates in control of normal coronary vascular tone. With VC108, coronary autoregulation was abolished and CBF became CDP dependent (r2=0.96, p=0.004). ConclusionGPR39 and its endogenous agonist 15-HETE together orchestrate coronary autoregulation when CDP is reduced. These novel findings provide a mechanism for coronary autoregulation and could direct pharmacological treatment of various coronary syndromes in humans.

BackgroundWingless/Int-1 (Wnts) proteins are canonical Frizzled receptor ligands. Recent evidence indicates that some Wnts, including Wnt9b and Wnt5a, bind to polycystin 1 (PKD1), a transmembrane protein which can couple to polycystin 2 (PKD2) to form a non-selective cation channel. The functional significance of Wnts binding to PKD1 is unclear. Here, we tested the hypothesis that Wnts act through PKD1/PKD2 channels on endothelial cells (ECs) to regulate arterial contractility and blood pressure and investigated the cellular source and secretory regulation of vasoactive Wnt proteins. MethodsA wide variety of approaches, including inducible EC-specific PKD1 and PKD2 knockout mice, reverse-transcription polymerase chain reaction, Western blotting, immunofluorescence, pressurized artery myography, blood pressure measurements, patch-clamp electrophysiology, in vivo and in vitro Wnt and nitric oxide assays, and Wnt secretion assays. ResultsIntravascular Wnt9b or Wnt5a administration stimulates an EC PKD1/PKD2-dependent dilation in pressurized resistance-size arteries. Wnt9b and Wnt5a are present in serum and plasma and intravenous infusion rapidly stimulates a blood pressure reduction which requires EC PKD1. Wnts stimulate a PKD1-dependent non-selective cation current in ECs which through Ca2+ signaling activates endothelial nitric oxide synthase (eNOS) and small conductance Ca2+-activated K+ channels to induce vasodilation. Wnt9b acts solely via PKD1/PKD2 channels, whereas Wnt5a stimulates signaling through PKD1/PKD2, Frizzled-7 (Fzd-7), Dishevelled and c-Jun N-terminal kinase (JNK). Intravascular flow stimulates angiotensin II type 1 (AT1) receptors, which through Gq/11 and Porcupine activate Wnt9b and Wnt5a secretion in ECs. Wnts secreted in response to flow activate PKD1/PKD2 signaling in ECs and contribute to flow-mediated vasodilation. ConclusionsIntravascular flow activates AT1 receptors, which through Gq/11 and Porcupine stimulate Wnt9b and Wnt5a secretion in ECs. Wnt9b activates PKD1/PKD2 channels whereas Wnt5a stimulates both PKD1/PKD2 and Fzd-7 in ECs to induce vasodilation. Wnts contribute to flow-mediated autocrine/paracrine dilation and reduce blood pressure. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/712518v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@158bad1org.highwire.dtl.DTLVardef@5113eforg.highwire.dtl.DTLVardef@f3b94eorg.highwire.dtl.DTLVardef@10ab479_HPS_FORMAT_FIGEXP M_FIG C_FIG

This study aimed to determine the impact of inborn metabolic fitness and early life exercise training on whole body and brown adipose tissue (BAT) energetics. We carried out comprehensive metabolic phenotyping on 4-week old rats bred for high (high-capacity runner, HCR) and low (low-capacity runner, LCR) running capacity following randomization to voluntary wheel running (VWR) or control (CRTL) for 6-weeks. High-resolution respirometry and untargeted proteomics were then employed to determine the impact of inborn fitness and early life exercise on BAT function. When accounting for differences in body mass, early life exercise (VWR) resulted in greater basal and total energy expenditure, irrespective of strain (P < 0.0001 for both). Both leak and uncoupling protein 1 (UCP1) dependent respiratory capacities in isolated BAT mitochondria were greater in rats randomized to VWR compared to CTRL in both HCR (P < 0.01) and LCR (P < 0.05) strains. Similarly, mitochondrial sensitivity to the UCP1 inhibitor GDP was greater in both HCR (P < 0.01) and LCR (P < 0.05) rats randomized to VWR versus control. The BAT proteome differed in CTRL HCR and LCR rats, were there was enrichment in proteins related to branched chain oxidation and mitochondrial fatty acid oxidation in HCR rats. VWR remodeled the BAT proteome, where 151 proteins were differentially expressed in LCR BAT and 209 differentially expressed in LCR BAT following VWR. In both stains, there was an enrichment in proteins related to metabolism mitochondrial function in response to VWR. However, when comparing strains, 39 proteins were differentially expressed in BAT in HCR rats compared to LCR rats in response to VWR. These proteins were related to carboxylic acid and amino acid metabolism. Collectively, inborn fitness impacts body mass and composition, exercise behaviors, and the BAT proteome in early life. Early life exercise alters whole body and BAT energetics irrespective of inborn fitness, augmenting basal and total energy expenditure and BAT thermogenic capacity and function.

Metabolic syndrome (MetS), characterized by abdominal obesity, insulin resistance, dyslipidemia, and hypertension, affects a substantial proportion of the global population and increases the risk for cardiovascular disease, diabetes, and metabolic dysfunction-associated steatotic liver disease (MASLD). Despite its prevalence, there are currently no effective pharmacological therapies targeting MetS, highlighting the need to identify novel etiological mechanisms, particularly within the gastrointestinal (GI) tract. Using a mouse model of MetS and healthy lean controls, we assessed the colonic microenvironment through metabolomic, transcriptomic, and microbiome analyses. Colonic organoids were cultured to further explore epithelial alterations. Additionally, human MetS fecal metabolomics data were cross-compared with the mouse model to validate translational relevance. MetS mice exhibited upregulation of colonic anabolic pathways, including glycolysis, the pentose phosphate pathway, and the tryptophan/kynurenine pathway, without evidence of intestinal inflammation. Microbiome analysis revealed an increased abundance of the genus Lactobacillus in MS NASH mice. Colonic organoids from MetS mice showed altered goblet cell differentiation. Comparative analysis with human MetS fecal metabolomics demonstrated similar dysregulated pathways, underscoring the translational relevance of these findings. Our study reveals significant metabolic and microbial alterations in the colon of MS NASH mice, implicating a dysfunctional GI tract as a potential etiological factor in MetS. These findings highlight specific metabolic pathways and microbial signatures that could serve as future therapeutic targets for MetS. NEW & NOTEWORTHYThis study identifies the colon as a metabolically active tissue affected in metabolic syndrome. Despite the absence of intestinal inflammation, MS NASH mice displayed altered colonic metabolism and microbiota composition, with conserved metabolite changes matching those seen in humans with metabolic syndrome. These findings highlight colonic metabolic dysfunction as a potential driver of gut dysbiosis and disease progression in metabolic syndrome and MASLD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/716131v1_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@1b7c685org.highwire.dtl.DTLVardef@4a832aorg.highwire.dtl.DTLVardef@1e95c66org.highwire.dtl.DTLVardef@1b14209_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundActivated platelets release polyphosphate (polyP), a linear polymer of inorganic phosphate residues, from dense granules. Experiments performed under no-flow conditions show that polyP alters the kinetics of tissue factor (TF) pathway reactions, accelerating FXI activation by thrombin and FV activation by FXa and thrombin, and may impact inhibition by tissue factor pathway inhibitor (TFPI). How polyP influences this pathway in conjunction with platelet deposition under flow remains understudied. ObjectivesTo investigate how polyP-mediated acceleration of FV and FXI activation modulates thrombin generation under flow in TF-initiated coagulation. MethodsWe extended a previously validated mathematical model of platelet deposition and coagulation under flow to examine polyP-mediated effects following a small vascular injury during intravascular clotting. Simulations varied the surface density of TF exposed, wall shear rate, and plasma TFPI concentration. ResultsPolyP shifts the threshold TF density for a thrombin burst to lower TF densities. For TF densities above this threshold, polyP shortens the lag time to thrombin generation in a TF- and shear-rate-dependent manner. Although no explicit effect of polyP on TFPI function was included in the model, thrombin generation was much less sensitive to TFPI concentration with polyP, in a TF-dependent manner. Relative contributions of accelerations of FV and FXI activations depend on incompletely known enhancements by polyP. ConclusionsThe experimentally observed influence of polyP on TFPI function depends on TF density and may arise indirectly from accelerated FV and FXI activation, with the dominant effect arising through accelerated thrombin-mediated conversion of FV to FVa.

Endurance (END) or resistance exercise (RE) training results in adaptations that give rise to distinct skeletal muscle phenotypes. Hallmarks of RE include increases in muscle fibre and muscle cross-sectional area and strength, whereas END increases mitochondrial content. Such distinct phenotypes arise from differential metabolic and mechanical signal transduction, transcriptional, and protein translation pathways, culminating in exercise mode-specific adaptations in the muscle proteome. However, little empirical data exist on the protein-specific dynamic responses underlying training-mode-specific adaptations in humans. Using a model of unilateral exercise combined with stable isotope labelling with deuterium oxide, we measured changes in synthesis and abundance from baseline and during early (week 1) and later (week 10) periods of adaptation to END and RE training in young healthy adults (n = 14; 8 female, 6 male; 20 {+/-} 1 y, 70 {+/-} 10 kg). We quantified changes in the abundance (n = 1146 proteins) and synthesis (n = 247 proteins) profiles of skeletal muscle across a 5-day pre-training baseline period and during early and later adaptation to RE and END. Abundance profiling revealed mode-specific proteome remodelling, whereby RE increased ribosomal and contractile protein networks, whereas END increased mitochondrial inner membrane proteins after 10 weeks of training. The protein-specific synthesis rates of 119 proteins showed training-induced differences (P < 0.1 and log2 fold change > 1), including subsets of structural proteins that responded differently to RE and END training modes. Notably, distinct Z-disc proteins, such as XIRP1 (RE-specific) and LDB3 (END-specific), exhibited mode-specific regulation despite sharing a similar subcellular localisation. We report, for the first time, that divergent phenotypic adaptations to RE and END extend beyond changes in bulk fraction-specific synthesis rates and are regulated by training-mode-specific adaptations in distinct protein subsets within similar subcellular protein locations.

BackgroundCarnitine plays an obligatory role in energetics owing to its role in the translocation of long-chain fatty acids into the mitochondrion for oxidation. Here, we determined the metabolic and behavioral consequences of systemic carnitine deficiency (SCD) in mice. MethodsFemale C57BL/6J mice were randomized to receive normal drinking water (control, n = 8) or drinking water supplemented with mildronate 4g.L-1 (mildronate, n = 8) for 21 days. Body composition was assessed at baseline and post treatment. Metabolic and behavioral phenotyping was performed continuously over 72 hours following 14 days of control or mildronate treatment. Stable isotope were used to assess whole-body substrate oxidation. Carnitine subfractions were quantified in skeletal muscle and liver, as was mitochondrial respiratory function. Liver and muscle samples also underwent proteomic analysis. ResultsMildronate treatment depleted total carnitine in muscle and liver by [~]97% (P < 0.001) and [~]90% (P < 0.001), respectively. Carnitine depletion was accompanied by lower total energy expenditure (P = 0.01), attributable to lower voluntary wheel running (P = 0.01). Oxidation rates of palmitate (P < 0.01) but not octanoate were lower whereas rates of glucose oxidation were greater in carnitine depleted mice (P < 0.01). Mitochondrial respiratory capacity was unaltered by carnitine deficiency. Carnitine deficiency remodeled muscle and liver proteomes to support lipid oxidation and energy production. SummaryIn mice, carnitine deficiency is characterized by decreased long-chain fatty acid oxidation despite preserved mitochondrial respiratory capacity. Carnitine deficiency resulted in lower voluntary exercise and a concomitant reduction in energy expenditure.

Right ventricular failure (RVF) is a serious disease with a high mortality but no effective pharmacologic treatments. We reported RVF was reversed by chronic treatment with an 1A-adrenergic receptor (1A-AR) agonist. Recent studies suggest mitochondrial dysfunction contributes to RVF. Therefore, we investigated if reversal of RVF by chronic 1A-AR agonist treatment involved improved mitochondrial function. A mouse model of RVF caused by pulmonary artery constriction (PAC) for 2 wk was chronically treated for a further 2 wk. with a low dose of the 1A-AR agonist A61603 (10 ng/kg/day) or vehicle (no drug control). RV dysfunction was assessed from the fractional shortening of the RV outflow tract (RVOT FS). RVOT FS for sham controls (46.5 {+/-} 1.3 %, n = 9) was reduced 4 wk after PAC (27.6 {+/-} 1.5 %, n = 13, P < 0.0001), but was higher after PAC plus 2 wk A61603 treatment (34.5 {+/-} 0.6 %, n = 14, P < 0.001). RV myocardial respiration rate (O2 consumption) for sham controls (776 {+/-} 51 pM/s/mg, n = 9) was reduced 4 wk after PAC (493 {+/-} 28 pM/s/mg, n = 15, P <0.0001), but was higher after PAC plus 2 wk A61603 treatment (634 {+/-} 30 pM/s/mg, n = 11, P <0.05). RV myocardial ATP level for sham controls (3.3 {+/-} 0.1 mM, n = 10) was reduced 4 wk after PAC (1.9 {+/-} 0.1 mM, n = 6, P < 0.0001), but was higher after PAC plus 2 wk A61603 treatment (2.6 {+/-} 0.13 mM, n = 7, P < 0.01). In conclusion, reversal of RVF after chronic A61603 treatment involved reversal of mitochondrial dysfunction. Consistent with our previous studies, this study suggests that the 1A-AR is a therapeutic target to treat RVF. HighlightsRV failure is reported to involve mitochondrial dysfunction which might impair RV contraction by decreasing cardiomyocyte ATP level. Using the pulmonary artery constriction model of RV failure, we found that chronic treatment with an 1A-adrenergic receptor agonist increased RV myocardial respiration rate, increased RV myocardial ATP level, and increased RV function. These findings suggest that the 1A-adrenergic receptor is a therapeutic target for treating RV failure, and that the mechanism involves improved RV cardiomyocyte bioenergetic status.

Exercise is a cornerstone therapy for diabetes because working skeletal muscles take up glucose at dramatically greater rates than postprandial insulin-stimulated glucose uptake and, notably, do so without a requirement for insulin. This remarkable ability of working muscles is preserved in diabetes, when muscles become resistant to insulin. However, the mechanism of insulin-independent glucose uptake by working muscles is not fully understood. Here we describe a previously unrecognized glucose uptake pathway in muscle, which we refer to as "mSGLT" based on shared properties with the Sodium Glucose Linked Transporter family. In contrast to the abundant GLUT4 transporter, mSGLT is not regulated by insulin, requires Na,K-ATPase-2 activity, and transports the hexose -methyl-D-glucoside (MDG), a glucose derivative that is handled by SGLTs but not GLUT4. The mSGLT pathway and GLUT transport pathways are independent and additive. In addition to exercise, mSGLT imports glucose under other conditions of adrenergic stimulation, which inhibits pancreatic insulin release and reduces the insulin sensitivity of muscle. SGLT2-specific antibodies recognize a protein in muscle of similar size to the kidney SGLT2; this protein localizes to the muscle t-tubules, together with Na,K-ATPase-2 and MAP17, the regulatory subunit of SGLT2. However, skeletal muscles do not express a full-length transcript of Slc5a2 (SGLT2), and SGLT2-specific inhibitors do not inhibit mSGLT with high affinity. The novel transporter may be a muscle variant of Slc5a2 that results from post-transcriptional or post-translational mechanisms. mSGLT and its regulation offer potential muscle-specific therapeutic targets for treating hyperglycemia and other conditions when insulin-stimulated glucose disposal into muscle is impaired.

BackgroundAntihypertensive and cardioprotective medications are prescribed to pregnant women and include Ca2+ channel blockers (CCBs; amlodipine, nifedipine), - (doxazosin) and {beta}-(labetalol, bisoprolol, nebivolol) adrenergic receptor antagonists, and -adrenergic receptor agonists (methyldopa). These vasoactive drugs enter the fetal circulation, with unknown effects on the fetoplacental vasculature. We aimed to investigate whether cardiovascular medications modulate human fetoplacental vascular tone, which may impair or enhance placental perfusion. MethodsChorionic plate arteries (CPAs) were obtained from the placentas of women with normotensive pregnancy (N=28), with unmedicated hypertension (N=14), and those chronically medicated (N=61) with either amlodipine, nifedipine, labetalol or bisoprolol, or a combination of CCBs and labetalol. Using wire myography, ex vivo effects of amlodipine, nifedipine, labetalol, methyldopa, doxazosin, bisoprolol and nebivolol were tested in a concentration-dependent manner (10-11-10-5M) in pre-constricted CPAs isolated from the placentas of normotensive women. Differences in CPA vascular reactivity in response to chronic exposure to hypertension and/or cardiovascular medications was assessed by vasoconstriction to high potassium physiological solution (KPSS; 120mM) and to the thromboxane A2 mimetic (U46619; 10-10-2x10-6M), and relaxation to the nitric oxide donor, sodium nitroprusside (SNP; 10-10-10-5M). ResultsIn pre-constricted CPAs isolated from normotensive women, acute exposure to amlodipine, nifedipine, doxazosin and nebivolol promoted significant vasorelaxation (P<0.05). CPAs acutely exposed to labetalol, methyldopa (P<0.05) and bisoprolol (P<0.001) exhibited increased vasoconstriction compared to their respective diluent controls. CPAs from women with chronic hypertension and from those who had chronic labetalol treatment exhibited significantly reduced vasoconstriction to KPSS (P<0.05). CPAs from women with chronic hypertension and exposure to bisoprolol also had significantly attenuated vascular responses to U46619 and SNP (P<0.01 and P<0.01, respectively), compared to normal pregnancy. ConclusionsMaternal hypertension impairs vascular responses of the placenta. Cardiovascular medications prescribed during pregnancy may dysregulate placental vascular function. Further research is warranted to evaluate the relative safety of cardiovascular medications in pregnancy, as their distinct effects on fetoplacental vascular function may have important implications for maternal and fetal outcomes. Mechanistic studies alongside clinical correlations are essential to guide evidence-based prescribing.

BackgroundPreeclampsia, a maternal hypertensive syndrome affect fetal brain development and cerebral angiogenesis, with potential acute and long-term consequences. Underlying mechanisms of these brain vascular alterations are unknown. This study investigates the role of thrombospondin-1 (TSP-1), an antiangiogenic glycoprotein, as a key mediator of communication between the fetoplacental and fetal brain endothelium in the context of preeclampsia. MethodsConditioned media (CM) of human umbilical vein endothelial cells (HUVECs) from normal pregnancies (NP-CM) and preeclamptic pregnancies (PE-CM), were used to treat human (hCMEC/D3) and murine brain microvascular endothelial cells (BMECs). A proteomic analysis was performed in plasma of the umbilical cord of normal pregnancy and preeclampsia. TSP-1 was identify using proteomic analysis and confirmed by Western blot. PE-CM depleted of TSP-1, using immunoprecipitation, was used to evaluate protein-protein interaction with vascular endothelial growth factor (VEGF). Antibody-mediated blockage of TSP-1 was used to investigate antiangiogenic effect and pro-angiogenic signaling pathways in brain endothelial cells exposed to PE-CM. ResultsPE-CM significantly reduced angiogenesis, migration, and invasion of brain endothelial cells and altered cytoskeletal organization. These effects were accompanied by reduced VEGFR2 and AKT signaling, indicating impaired angiogenic pathways. Proteomic analysis of umbilical cord plasma revealed elevated TSP-1 levels in preeclampsia, which was confirmed by Western blotting. TSP-1 was also increased in PE-CM, and immunoprecipitation assays suggested a protein-protein interaction with VEGF. Antibody-mediated blockade of TSP-1 restored angiogenesis, as reflected by increased total tube length, and rescued VEGFR2 and AKT signaling in brain endothelial cells exposed to PE-CM. ConclusionTSP-1-mediated endothelium-endothelium communication between placenta-brain axis in offspring of mothers with preeclampsia. This communication mediated by TSP-1 may contribute to acute and long-lasting cerebrovascular dysfunction observed in infants exposed to preeclampsia.

Osteoblasts generate bone by secreting collagen and mineralizing it in response to various signaling cues. We have previously shown that the majority of ATP generated by differentiated osteoblasts in response to glucose is through glycolysis in contrast to undifferentiated cells that are more dependent on oxidative phosphorylation. To confirm our previous findings, metabolomics was performed for unlabeled polar metabolites, revealing elevated glycolytic metabolites at the later stages of differentiation. Krebs cycle (TCA cycle) metabolites were also changed confirming metabolic rerouting with differentiation. We hypothesized that an increase in mitophagy shifts ATP generation towards glycolysis resulting in the observed bioenergetic and metabolic changes. Utilizing calvarial osteoblasts isolated from a mitophagy reporter mouse model (MitoQC), an increase in mitophagy and the mitophagy receptor, Bnip3, was observed with osteoblast differentiation. KD of Bnip3 in osteoblasts inhibited differentiation and mineralization arising from impaired mitochondrial function. In vivo, male Bnip3 null mice exhibited a significant decrease in osteoblast numbers resulting in lower bone mass. Mechanistically we identified decreased fusion and increased fission factors, impaired stress signaling and increased proapoptotic factors in the absence of Bnip3. These data demonstrate for the first time that BNIP3 expression and mitophagy during osteoblast differentiation are necessary for relieving mitochondrial stress to maintain optimal bone mass.

ObjectiveTandem cycling requires a coordinated effort between the pilot and the stoker. Previous research suggests that randomly paired tandem cyclists produce lower power output than when cycling solo. This study examined how a cyclists individual ability and their position on the tandem (pilot or stoker) affects pair performance, when partners are either closely matched or differ substantially in solo cycling capacity, as this might be relevant for training and selection. MethodsTwenty-three trained cyclists completed three 10-minute time trials: solo, equal-capacity tandem ([&le;]25 W difference in solo performance), and unequal-capacity tandem ([&ge;]40 W difference). Mean power output, heart rate, cadence, and rating of perceived exertion (RPE) were recorded. Positions (pilot or stoker) were counterbalanced. Linear mixed-effects models assessed effects of capacity and position. ResultsRelative to solo cycling, equal-capacity tandem pairs revealed lower power output (-3.9%), lower heart rate (-2.3%), and lower RPE (-11.5%). Unequal-capacity tandems differed from solo only in heart rate (-2.7%). Stokers produced lower power relative to solo (-5.3%) and relative to pilots (-3.7%) and reported lower RPE relative to solo (-13.9%), while pilots matched their solo power at a lower heart rate (-2.9%). Cadence did not differ across conditions. Total tandem power averaged 95.6% of combined solo power, and differences in partner capacity did not significantly affect combined power output. ConclusionThis study provides the first known experimental data on how partner matching affects individual and combined power output in tandem cycling. Equal- and unequal-capacity tandem pairs showed similar performance. Lower power and RPE among stokers suggest reduced engagement or a redistribution of effort between riders. These findings highlight that effective tandem performance depends on physiological capacity and rider position on the tandem, but not on the difference in capacity between partners.

BACKGROUNDThe vascular endothelium is a dynamic tissue central to vascular homeostasis and disease, with endothelial cells (ECs) exhibiting plasticity that drives adaptive remodeling. Reelin, a secreted extracellular matrix glycoprotein critical for neuronal migration via ApoER2/VLDLR-DAB1 signaling, may also modulate vascular function and inflammation. However, its direct role in EC biology remains unclear. We investigated Reelin as a context-dependent signaling modulator in ECs, assessing its engagement of non-canonical pathways and regulation of endothelial plasticity relevant to cardiovascular pathology. METHODSHuman endothelial cells were stimulated with recombinant Reelin and analyzed by immunoblotting, immunofluorescence, and functional assays. Time-course studies assessed signaling, including phosphorylation of FAK, AKT, and DAB1 by Western blotting, while wound-healing assays quantified endothelial migratory capacity in vitro systems. RESULTSReelin rapidly robustly activated noncanonical signaling in endothelial cells, increasing FAK and AKT phosphorylation in a time-dependent manner consistent with cytoskeletal remodeling. Canonical DAB1 activation was limited. Functionally, Reelin enhanced migration, upregulated Endoglin/CD105, and induced a remodeling-associated phenotype. Reelin silencing altered endothelial phenotype, clearly indicating a role in homeostasis. Signaling was independent of VEGFR2 interaction. Overall, Reelin preferentially engages FAK/AKT pathways to drive partial phenotypic modulation without full endothelial-to-mesenchymal transition. CONCLUSIONWe show that Reelin is a previously unrecognized regulator of endothelial signaling and plasticity, acting via non-canonical FAK- and AKT-dependent pathways. By partially and dynamically modulating endothelial phenotype, Reelin promotes a remodeling-permissive state without triggering full mesenchymal transition. These findings identify Reelin as a novel modulator of endothelial function with potential implications for vascular remodeling and cardiovascular disease. What Are the Clinical Implications?Our findings identify Reelin as a modulator of endothelial signaling with a clear bias toward non-canonical FAK- and AKT-dependent pathways that regulate endothelial plasticity and remodeling. This signaling profile is highly relevant to vascular diseases in which endothelial dysfunction is driven by maladaptive cytoskeletal reorganization, altered migration, and persistent activation rather than complete loss of endothelial identity. The ability of Reelin to promote partial and dynamically regulated phenotypic modulation suggests that it may operate at early and potentially reversible stages of vascular pathology. In this context, dysregulated Reelin signaling could contribute to pathological vascular remodeling, including processes underlying atherosclerosis, fibrosis, and microvascular dysfunction. These results also raise the possibility that circulating or locally produced Reelin may serve as an indicator of endothelial activation state, providing a novel biomarker for vascular disease progression. Importantly, the identification of a signaling bias toward FAK- and AKT-dependent pathways highlights potential therapeutic targets downstream of Reelin that could be selectively modulated to limit maladaptive endothelial remodeling while preserving essential endothelial functions. Collectively, this study positions Reelin signaling as a previously unrecognized and potentially actionable pathway in the regulation of endothelial behavior, with direct implications for the development of targeted strategies aimed at preventing or attenuating cardiovascular disease progression

The use of decellularized diseased livers in regenerative medicine is a promising approach for eliminating organ shortages. Bioengineering studies have shown that ECM can impact cell physiology, inducing cell activation, function, and ECM deposition, which suggests that the ECM has a "memory" that is involved in the outcome after recellularization. However, the effect of diseased ECM memory on new cells in vitro and in vivo has not been thoroughly investigated. Since it has been increasingly recognized that liver ECM changes due to different factors, it is comprehensively that diseased ECM obtained from discarded organs will ensure a distinct environment and impact cell survival and physiology. Thus, we aimed at investigating the impact of the memory of diseased ECM obtained from metabolic dysfunction-associated steatohepatitis (MASH)-derived organs on steatohepatitis establishment. To address this aim, we explored decellularized ECM obtained from rats and humans with MASH in different contexts. First, MASH ECM was characterized and then submitted to transplantation to investigate whether a MASH-derived ECM could be used as a scaffold for transplantation and to promote steatohepatitis features in control animals. Histological analysis revealed that the MASH-ECM was completely recellularized after transplantation in both control and MASH recipient rats. However, steatosis and fibrosis were observed in MASH ECM after transplantation in both groups. Molecular analysis showed that MASH ECM stimulates de novo lipogenesis and fibrosis 30 days after transplantation. Untargeted metabolomic analysis revealed that cells grown on MASH ECM had a similar metabolic profile, even when transplanted into healthy or MASH recipient rats. In addition, we observed that MASH ECM promoted impaired lipid oxidation and mitochondrial dysfunction when transplanted into healthy recipients. Altered lipid turnover and inflammatory signaling were observed in MASH ECM transplanted in MASH recipients. In vitro analysis revealed that MASH ECM induced lipid accumulation in HepG2 cells after 10 days of culture. Calcium signalling experiments obtained from HepG2 cells cultured in MASH ECM showed a lower response to ATP, a reduced calcium signalling amplitude, and a distinct response profile than that observed in healthy ECM. On the other hand, a diseased human-derived ECM could still provide an environment that allows cell development. Taken together, our data showed that MASH ECM impacts cell metabolism, promoting steatohepatitis maintenance. In conclusion, our data confirm that diseased ECM memory can impact cell physiology contributing to disease progression.

Intervertebral disc degeneration is associated with loss of nucleus pulposus (NP) cell phenotype and extracellular matrix, both processes linked to changes in cytoskeletal contractility and cell shape. Here, we tested whether microenvironment-specific modulation of RhoA signaling can restore NP-like morphology and gene expression in NP cells cultured in 2D and in 3D alginate. In 2D monolayer culture, where cells are spread and mechanically activated, pharmacologic inhibition of RhoA with CT04 reduced RhoA activity, decreased actomyosin contractility gene expression, and shifted morphology toward a smaller, more circular phenotype. Bulk RNA sequencing showed that CT04 treatment increased expression of NP phenotypic and matrix-related genes including ACAN, GDF5, CHST3, and MUSTN1 while decreasing expression of catabolic and fibroblast-associated genes including ADAMTS1/9 and COL1, consistent with enrichment of extracellular matrix pathways. In contrast, RhoA activation with CN03 in 2D culture increased actin and phosphorylated myosin light chain intensity but produced limited phenotypic improvement. In 3D alginate, which minimizes integrin-mediated adhesion, baseline actomyosin markers were reduced relative to 2D culture. In alginate, RhoA activation with CN03 increased the amount of actin, phosphorylated myosin light chain, and actomyosin gene expression, yet also promoted a more compact, circular morphology and increased NP markers, including ACAN and KRT19 with repeated dosing. Across culture conditions, increased cell roundness was consistently associated with increased ACAN expression, indicating strong coupling between cytoskeletal state, morphology, and NP matrix programs. Together, these findings demonstrate that RhoA pathway perturbation can promote NP phenotypic gene expression in both 2D and 3D culture, but the direction of optimal modulation depends on the microenvironment, supporting RhoA signaling as a context-dependent therapeutic target for disc regeneration.

BackgroundSex-related differences in cardiovascular disease suggest the presence of intrinsic vasoprotective mechanisms, with estrogen recognized as an important modulator of endothelial function. Building on existing evidence, the present study provides mechanistic insights into how estrogen and nitric oxide (NO) signaling regulate selective pathways of oxLDL uptake, mitochondrial dynamics, and inflammatory responses during early atherogenesis. MethodsWe combined an in vitro endothelial cell-macrophage co-culture model with in vivo studies in low-density lipoprotein receptor-knockout (LDLr-/-) mice to investigate the role of estrogen in early atherosclerotic processes. Human aortic endothelial cells (HAECs) were exposed to oxidized low-density lipoprotein (oxLDL) in the presence or absence of 17{beta}-estradiol (E2) and the nitric oxide (NO*) donor S-nitroso-N-acetylcysteine (SNAC). Key outcomes included oxLDL uptake, mitochondrial oxidative stress, mitochondrial dynamics, and inflammatory signaling. In vivo, male and female LDLr-/- mice were exposed to a short-term high-fat diet with or without SNAC treatment. Plasma lipid levels, blood pressure, aortic lesion formation, and cardiac remodeling were evaluated. ResultsE2 reduced oxLDL uptake and oxidative stress, effects recapitulated by SNAC; however, these responses involved distinct entry pathways, with E2 preferentially modulating lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) dependent uptake and SNAC targeting caveolae-associated mechanisms. In parallel, both E2 and SNAC reduced Scavenger Receptor Class B Type 1 (SR-B1) expression, suggesting an additional modulation on oxLDL transcytosis via this mechanism. Endothelial cells exposed to oxLDL exhibited altered mitochondrial regulatory proteins, including superoxide dismutase 2 (SOD-2), dynamin-related protein 1 (Drp-1), and optic atrophy protein 1 (OPA-1). Despite reducing oxidative stress, E2 increased the expression of adhesion molecules and enhanced monocyte adhesion in response to oxLDL exposure, particularly when combined with SNAC. Strikingly, E2 also modulated macrophage responses, increasing interleukin receptor antagonist (IL-1ra) expression and reducing GDF15, macrophage inhibitory factor (MIF), macrophage inflammatory protein 3 alfa (MIP-3), and matrix metalloproteinase 9 (MMP-9) levels, consistent with a less pro-inflammatory macrophage profile. In vivo, HFD increased plasma lipid levels and atherosclerotic lesion area in LDLr-/- mice, whereas SNAC partially attenuated these effects without affecting plasma lipid levels. In vivo, female LDLr-/- mice developed approximately 50% smaller aortic lesions than males, despite comparable or higher plasma lipid levels. A dyslipidemia led to increased blood pressure and a hypertensive phenotype in both males and females. SNAC treatment reduced lesion burden in both sexes and prevented diet-induced hypertension in females. ConclusionEstrogen limits early atherogenic injury by reducing endothelial uptake of oxLDL, preserving mitochondrial homeostasis, and modulating inflammatory signaling. Together, the E2 and NO pathways regulate early atherosclerosis through distinct yet complementary mechanisms, offering a potential framework for vascular-protective strategies.

Mitochondrial membrane potential ({Delta}{Psi}m) is central to ATP production, ion homeostasis, and cell survival, reflecting the functional state of the inner mitochondrial membrane and oxidative phosphorylation. Accurate assessment of {Delta}{Psi}m is therefore essential for understanding mitochondrial physiology and dysfunction in health, ageing, and disease. Lipophilic cationic fluorescent dyes, such as TMRM and TMRE, are widely used to monitor {Delta}{Psi}m in live cells, enabling high-temporal-resolution imaging of both steady-state membrane potential and dynamic fluctuations. Beyond stable bioenergetic measurements, live-cell imaging reveals transient, reversible depolarisation events, known as mitochondrial "flickers." These events, observed across multiple cell types and imaging platforms, are often associated with brief openings of the mitochondrial permeability transition pore (mPTP) and may represent regulated mitochondrial excitability, rather than irreversible damage. While excessive or synchronised depolarisations may signal mitochondrial injury, transient flickers are increasingly viewed as potential signalling mechanisms within the mitochondrial network. This work discusses methodological considerations for {Delta}{Psi}m imaging, the biological significance of mitochondrial flickers, and the importance of distinguishing physiological events from probe- and light-induced artefacts, highlighting the emerging concept of mitochondria as dynamic and communicative bioenergetic networks.

Pregnant women are particularly susceptible to adverse outcomes from environmental heat, yet the physiological effects of acute heat exposure during pregnancy remain poorly understood. Some physiological changes are monitored in humans; however, investigation of underlying molecular mechanisms requires invasive methods that can only be ethically applied in mammalian models. Moreover, research with animal models has largely focused on early and lethal teratogenic effects of heat exposure and lacks longitudinal physiological monitoring, detailed parameterisation of heating regimes and in-depth investigation of underlying mechanisms. Here we used a mouse model to investigate the impact of a controlled acute heat exposure at mid-gestation (E12{middle dot}5), slowly elevating core body temperature (CBT) over 210mins to raise CBT by [~]1{degrees}C. Using high-frequency ultrasound and morphological analyses, we observed delayed alterations in placental and foetal cerebral blood flow indicative of a brain-sparing response, alongside reduced placental labyrinth zone size. Additionally, maternal cardiac function was impaired, accompanied by cardiac and renal fibrosis and elevated circulating soluble Flt-1 levels, an anti-angiogenic biomarker of gestational hypertension. These findings demonstrate that brief heat stress at mid-gestation can induce lasting effects on placental function and maternal cardiovascular health in a mammalian model, highlighting potential risks for pregnancy outcomes under increasing global temperatures. Together this data suggests that an acute exposure to heat elevating core body temperature by 1{middle dot}2{degrees}C can induce a long-term impact on both placenta and maternal health in a mouse model. It will be important to understand the molecular changes which underpin the pathophysiology and whether this is translated to humans.