Novel Role for Cardiolipin as a Target of Therapy to Mitigate Myocardial Injury Caused by Venoarterial Extracorporeal Membrane Oxygenation.

BACKGROUND: Cardiolipin is a mitochondrial-specific phospholipid that maintains integrity of the electron transport chain (ETC) and plays a central role in myocardial ischemia/reperfusion injury. Tafazzin is an enzyme that is required for cardiolipin maturation. Venoarterial extracorporeal membrane oxygenation (VA-ECMO) use to provide hemodynamic support for acute myocardial infarction has grown exponentially, is associated with poor outcomes, and is under active clinical investigation, yet the mechanistic effect of VA-ECMO on myocardial damage in acute myocardial infarction remains poorly understood. We hypothesized that VA-ECMO acutely depletes myocardial cardiolipin and exacerbates myocardial injury in acute myocardial infarction. METHODS: We examined cardiolipin and tafazzin levels in human subjects with heart failure and healthy swine exposed to VA-ECMO and used a swine model of closed-chest myocardial ischemia/reperfusion injury to evaluate the effect of VA-ECMO on cardiolipin expression, myocardial injury, and mitochondrial function. RESULTS: Cardiolipin and tafazzin levels are significantly reduced in the left ventricles of individuals requiring VA-ECMO compared with individuals without VA-ECMO before heart transplantation. Six hours of exposure to VA-ECMO also decreased left ventricular levels of cardiolipin and tafazzin in healthy swine compared with sham controls. To explore whether cardiolipin depletion by VA-ECMO increases infarct size, we performed left anterior descending artery occlusion for a total of 120 minutes followed by 180 minutes of reperfusion in adult swine in the presence and absence of MTP-131, an amphipathic molecule that interacts with cardiolipin to stabilize the inner mitochondrial membrane. Compared with reperfusion alone, VA-ECMO activation beginning after 90 minutes of left anterior descending artery occlusion increased infarct size (36±8% versus 48±7%; P<0.001). VA-ECMO also decreased cardiolipin and tafazzin levels, disrupted mitochondrial integrity, reduced electron transport chain function, and promoted oxidative stress. Compared with reperfusion alone or VA-ECMO before reperfusion, delivery of MTP-131 before VA-ECMO activation reduced infarct size (22±8%; P=0.03 versus reperfusion alone and P<0.001 versus VA-ECMO alone). MTP-131 restored cardiolipin and tafazzin levels, stabilized mitochondrial function, and reduced oxidative stress in the left ventricle. CONCLUSIONS: We identified a novel mechanism by which VA-ECMO promotes myocardial injury and further identify cardiolipin as an important target of therapy to reduce infarct size and to preserve mitochondrial function in the setting of VA-ECMO for acute myocardial infarction.

Bone Morphogenetic Protein 9 Reduces Cardiac Fibrosis and Improves Cardiac Function in Heart Failure.

BACKGROUND: Heart failure is a growing cause of morbidity and mortality worldwide. Transforming growth factor beta (TGF-ß1) promotes cardiac fibrosis, but also activates counterregulatory pathways that serve to regulate TGF-ß1 activity in heart failure. Bone morphogenetic protein 9 (BMP9) is a member of the TGFß family of cytokines and signals via the downstream effector protein Smad1. Endoglin is a TGFß coreceptor that promotes TGF-ß1 signaling via Smad3 and binds BMP9 with high affinity. We hypothesized that BMP9 limits cardiac fibrosis by activating Smad1 and attenuating Smad3, and, furthermore, that neutralizing endoglin activity promotes BMP9 activity. METHODS: We examined BMP9 expression and signaling in human cardiac fibroblasts and human subjects with heart failure. We used the transverse aortic constriction-induced model of heart failure to evaluate the functional effect of BMP9 signaling on cardiac remodeling. RESULTS: BMP9 expression is increased in the circulation and left ventricle (LV) of human subjects with heart failure and is expressed by cardiac fibroblasts. Next, we observed that BMP9 attenuates type I collagen synthesis in human cardiac fibroblasts using recombinant human BMP9 and a small interfering RNA approach. In BMP9-/- mice subjected to transverse aortic constriction, loss of BMP9 activity promotes cardiac fibrosis, impairs LV function, and increases LV levels of phosphorylated Smad3 (pSmad3), not pSmad1. In contrast, treatment of wild-type mice subjected to transverse aortic constriction with recombinant BMP9 limits progression of cardiac fibrosis, improves LV function, enhances myocardial capillary density, and increases LV levels of pSmad1, not pSmad3 in comparison with vehicle-treated controls. Because endoglin binds BMP9 with high affinity, we explored the effect of reduced endoglin activity on BMP9 activity. Neutralizing endoglin activity in human cardiac fibroblasts or in wild-type mice subjected to transverse aortic constriction-induced heart failure limits collagen production, increases BMP9 protein levels, and increases levels of pSmad1, not pSmad3. CONCLUSIONS: Our results identify a novel functional role for BMP9 as an endogenous inhibitor of cardiac fibrosis attributable to LV pressure overload and further show that treatment with either recombinant BMP9 or disruption of endoglin activity promotes BMP9 activity and limits cardiac fibrosis in heart failure, thereby providing potentially novel therapeutic approaches for patients with heart failure.

Mixed lineage kinase-3 prevents cardiac dysfunction and structural remodeling with pressure overload.

Myocardial hypertrophy is an independent risk factor for heart failure (HF), yet the mechanisms underlying pathological cardiomyocyte growth are incompletely understood. The c-Jun NH2-terminal kinase (JNK) signaling cascade modulates cardiac hypertrophic remodeling, but the upstream factors regulating myocardial JNK activity remain unclear. In this study, we sought to identify JNK-activating molecules as novel regulators of cardiac remodeling in HF. We investigated mixed lineage kinase-3 (MLK3), a master regulator of upstream JNK-activating kinases, whose role in the remodeling process had not previously been studied. We observed increased MLK3 protein expression in myocardium from patients with nonischemic and hypertrophic cardiomyopathy and in hearts of mice subjected to transverse aortic constriction (TAC). Mice with genetic deletion of MLK3 (MLK3-/-) exhibited baseline cardiac hypertrophy with preserved cardiac function. MLK3-/- mice subjected to chronic left ventricular (LV) pressure overload (TAC, 4 wk) developed worsened cardiac dysfunction and increased LV chamber size compared with MLK3+/+ littermates ( n = 8). LV mass, pathological markers of hypertrophy ( Nppa, Nppb), and cardiomyocyte size were elevated in MLK3-/- TAC hearts. Phosphorylation of JNK, but not other MAPK pathways, was selectively impaired in MLK3-/- TAC hearts. In adult rat cardiomyocytes, pharmacological MLK3 kinase inhibition using URMC-099 blocked JNK phosphorylation induced by neurohormonal agents and oxidants. Sustained URMC-099 exposure induced cardiomyocyte hypertrophy. These data demonstrate that MLK3 prevents adverse cardiac remodeling in the setting of pressure overload. Mechanistically, MLK3 activates JNK, which in turn opposes cardiomyocyte hypertrophy. These results support modulation of MLK3 as a potential therapeutic approach in HF. NEW & NOTEWORTHY Here, we identified a role for mixed lineage kinase-3 (MLK3) as a novel antihypertrophic and antiremodeling molecule in response to cardiac pressure overload. MLK3 regulates phosphorylation of the stress-responsive JNK kinase in response to pressure overload and in cultured cardiomyocytes stimulated with hypertrophic agonists and oxidants. This study reveals MLK3-JNK signaling as a novel cardioprotective signaling axis in the setting of pressure overload.

Conditional knockout of activin like kinase-1 (ALK-1) leads to heart failure without maladaptive remodeling.

Activin like kinase-1 (AlK-1) mediates signaling via the transforming growth factor beta (TGFß) family of ligands. AlK-1 activity promotes endothelial proliferation and migration. Reduced AlK-1 activity is associated with arteriovenous malformations. No studies have examined the effect of global AlK-1 deletion on indices of cardiac remodeling. We hypothesized that reduced levels of AlK-1 promote maladaptive cardiac remodeling. To test this hypothesis, we employed AlK-1 conditional knockout mice (cKO) harboring the ROSA26-CreER knock-in allele, whereby a single dose of intraperitoneal tamoxifen triggered ubiquitous Cre recombinase-mediated excision of floxed AlK-1 alleles. Tamoxifen treated wild-type (WT-TAM; n = 5) and vehicle treated AlK-1-cKO mice (cKO-CON; n = 5) served as controls for tamoxifen treated AlK-1-cKO mice (cKO-TAM; n = 15). AlK-1 cKO-TAM mice demonstrated reduced 14-day survival compared to cKO-CON controls (13 vs 100%, respectively, p < 0.01). Seven days after treatment, cKO-TAM mice exhibited reduced left ventricular (LV) fractional shortening, progressive LV dilation, and gastrointestinal bleeding. After 14 days total body mass was reduced, but LV and lung mass increased in cKO-TAM not cKO-CON mice. Peak LV systolic pressure, contractility, and arterial elastance were reduced, but LV end-diastolic pressure and stroke volume were increased in cKO-TAM, not cKO-CON mice. LV AlK-1 mRNA levels were reduced in cKO-TAM, not cKO-CON mice. LV levels of other TGFß-family ligands and receptors (AlK5, TBRII, BMPRII, Endoglin, BMP7, BMP9, and TGFß1) were unchanged between groups. Cardiomyocyte area and LV levels of BNP were increased in cKO-TAM mice, but LV levels of ß-MHC and SERCA were unchanged. No increase in markers of cardiac fibrosis, Type I collagen, CTGF, or PAI-1, were observed between groups. No differences were observed for any variable studied between cKO-CON and WT-TAM mice. Global deletion of AlK-1 is associated with the development of high output heart failure without maladaptive remodeling. Future studies exploring the functional role of AlK-1 in cardiac remodeling independent of systemic AVMs are required.

Circulating multimarker profile of patients with symptomatic heart failure supports enhanced fibrotic degradation and decreased angiogenesis.

BACKGROUND: Heart failure (HF) involves myocardial fibrosis and dysregulated angiogenesis. OBJECTIVE: We explored whether biomarkers of fibrosis and angiogenesis correlate with HF severity. METHODS: Biomarkers of fibrosis [procollagen types I and III (PIP and P3NP), carboxyterminal-telopeptide of type I collagen (ICTP), matrix metalloproteases (MMP2 and MMP9), tissue inhibitor of MMP1 (TIMP1)]; and angiogenesis [placental growth factor (PGF), vascular endothelial growth factor (VEGF), soluble Fms-like tyrosine kinase-1 (sFlt1)] were measured in 52 HF patients and 19 controls. RESULTS: P3NP, ICTP, MMP2, TIMP1, PGF, and sFlt1 levels were elevated in HF, while PIP/ICTP, PGF/sFlt1, and VEGF/sFlt1 ratios were reduced. PIP/ICTP, MMP-9/TIMP1, and VEGF/sFlt1 ratios were lowest among patients with severe HF. CONCLUSIONS: Severe HF is associated with collagen breakdown and reduced angiogenesis. A multimarker approach may guide therapeutic targeting of fibrosis and angiogenesis in HF.

Mechanically unloading the left ventricle before coronary reperfusion reduces left ventricular wall stress and myocardial infarct size.

BACKGROUND: Ischemia/reperfusion injury worsens infarct size, a major determinant of morbidity and mortality after acute myocardial infarction (MI). We tested the hypothesis that reducing left ventricular wall stress with a percutaneous left atrial-to-femoral artery centrifugal bypass system while delaying coronary reperfusion limits myocardial injury in a model of acute MI. METHODS AND RESULTS: MI was induced by balloon occlusion of the left anterior descending artery in adult male swine. In the MI group (n=4), 120 minutes of left anterior descending artery occlusion was followed by 120 minutes of reperfusion without mechanical support. In the mechanically supported group (MI+unload; n=4), percutaneous left atrial-to-femoral artery bypass was initiated after 120 minutes of ischemia, and left anterior descending artery occlusion was prolonged for an additional 30 minutes, followed by 120 minutes of reperfusion with device support. All animals were euthanized after reperfusion, and infarct size was quantified by triphenyltetrazolium chloride staining. Compared with baseline, mean left ventricular wall stress and stroke work were not changed at any point in the MI group but were decreased after reperfusion in the MI+unload group (mean left ventricular wall stress, 44 658 versus 22 963 dynes/cm(2); stroke work, 2823 versus 655 mm Hg·mL, MI versus MI+unload). Phosphorylation of reperfusion injury salvage kinase pathway proteins from noninfarcted left ventricular tissue was unchanged in the MI group but was increased in the MI+unload group. Compared with the MI group, total infarct size was reduced in the MI+unload group (49% versus 28%, MI versus MI+unload). CONCLUSIONS: These data support that first unloading the left ventricle despite delaying coronary reperfusion during an acute MI reduces myocardial injury.

Predicting mine water inflow volumes using a decomposition-optimization algorithm-machine learning approach.

Disasters caused by mine water inflows significantly threaten the safety of coal mining operations. Deep mining complicates the acquisition of hydrogeological parameters, the mechanics of water inrush, and the prediction of sudden changes in mine water inflow. Traditional models and singular machine learning approaches often fail to accurately forecast abrupt shifts in mine water inflows. This study introduces a novel coupled decomposition-optimization-deep learning model that integrates Complete Ensemble Empirical Mode Decomposition with Adaptive Noise (CEEMDAN), Northern Goshawk Optimization (NGO), and Long Short-Term Memory (LSTM) networks. We evaluate three types of mine water inflow forecasting methods: a singular time series prediction model, a decomposition-prediction coupled model, and a decomposition-optimization-prediction coupled model, assessing their ability to capture sudden changes in data trends and their prediction accuracy. Results show that the singular prediction model is optimal with a sliding input step of 3 and a maximum of 400 epochs. Compared to the CEEMDAN-LSTM model, the CEEMDAN-NGO-LSTM model demonstrates superior performance in predicting local extreme shifts in mine water inflow volumes. Specifically, the CEEMDAN-NGO-LSTM model achieves scores of 96.578 in MAE, 1.471% in MAPE, 122.143 in RMSE, and 0.958 in NSE, representing average performance improvements of 44.950% and 19.400% over the LSTM model and CEEMDAN-LSTM model, respectively. Additionally, this model provides the most accurate predictions of mine water inflow volumes over the next five days. Therefore, the decomposition-optimization-prediction coupled model presents a novel technical solution for the safety monitoring of smart mines, offering significant theoretical and practical value for ensuring safe mining operations.

Circulating Proteome Analysis Identifies Reduced Inflammation After Initiation of Hemodynamic Support with Either Veno-Arterial Extracorporeal Membrane Oxygenation or Impella in Patients with Cardiogenic Shock.

In-hospital mortality associated with cardiogenic shock (CS) remains high despite the use of percutaneous assist devices. We sought to determine whether support with VA-ECMO or Impella in patients with CS alters specific components of the plasma proteome. Plasma samples were collected before device implantation and 72 h after initiation of support in 11 CS patients receiving ECMO or Impella. SOMAscan was used to detect 1305 circulating proteins. Sixty-seven proteins were changed after ECMO (18 upregulated and 49 downregulated, p < 0.05), 38 after Impella (10 upregulated and 28 downregulated, p < 0.05), and only eight proteins were commonly affected. Despite minimal protein overlap, both devices were associated with markers of reduced inflammation and increased apoptosis of inflammatory cells. In summary, ECMO and Impella are associated with reduced expression of inflammatory markers and increased markers of inflammatory cell death. These circulating proteins may serve as novel targets of therapy or biomarkers to tailor AMCS use.

Reduced endoglin activity limits cardiac fibrosis and improves survival in heart failure.

BACKGROUND: Heart failure is a major cause of morbidity and mortality worldwide. The ubiquitously expressed cytokine transforming growth factor-ß1 (TGFß1) promotes cardiac fibrosis, an important component of progressive heart failure. Membrane-associated endoglin is a coreceptor for TGFß1 signaling and has been studied in vascular remodeling and preeclampsia. We hypothesized that reduced endoglin expression may limit cardiac fibrosis in heart failure. METHODS AND RESULTS: We first report that endoglin expression is increased in the left ventricle of human subjects with heart failure and determined that endoglin is required for TGFß1 signaling in human cardiac fibroblasts using neutralizing antibodies and an siRNA approach. We further identified that reduced endoglin expression attenuates cardiac fibrosis, preserves left ventricular function, and improves survival in a mouse model of pressure-overload-induced heart failure. Prior studies have shown that the extracellular domain of endoglin can be cleaved and released into the circulation as soluble endoglin, which disrupts TGFß1 signaling in endothelium. We now demonstrate that soluble endoglin limits TGFß1 signaling and type I collagen synthesis in cardiac fibroblasts and further show that soluble endoglin treatment attenuates cardiac fibrosis in an in vivo model of heart failure. CONCLUSION: Our results identify endoglin as a critical component of TGFß1 signaling in the cardiac fibroblast and show that targeting endoglin attenuates cardiac fibrosis, thereby providing a potentially novel therapeutic approach for individuals with heart failure.

Safety and efficacy of fecal microbiota transplantation in the treatment of graft-versus-host disease.

This article evaluates the efficacy and safety of FMT in the treatment of GVHD after HSCT using a systematic literature search to conduct a meta-analysis constructed of studies involving GVHD patients treated with FMT. 23 studies were included, among which 2 prospective cohort studies, 10 prospective single arm studies, 2 retrospective single arm studies, 2 case series and 7 case reports, comprise a total of 242 patients with steroid-resistant or steroid-dependent GVHD secondary to HSCT who were treated with FMT. 100 cases achieved complete responses, while 61 cases showed partial responses, and 81 cases presented no effect after FMT treatment. The estimate of clinical remission odds ratio was 5.51 (95% CI 1.49-20.35) in cohort studies, and the pooled clinical remission rate is 64% (51-77%) in prospective single arm studies and 81% (62-95%) in retrospective studies, case series and case reports. Five (2.1%) patients had FMT-related infection events, but all recovered after treatment. Other adverse effects were mild and acceptable. Microbiota diversity and composition, donor type, and other related issues were also analyzed. The data proves that FMT is a promising treatment modality of GVHD, but further validation of its safety and efficacy is still needed with prospective control studies.Clinical trial registration: Registered in https://www.crd.york.ac.uk/PROSPERO/ CRD42022296288.

ALK1 Deficiency Impairs the Wound-Healing Process and Increases Mortality in Murine Model of Myocardial Infarction.

The functional role of TGFß type I receptor, activin-like kinase (ALK)-1 in post-myocardial infarction (MI) cardiac remodeling is unknown. We hypothesize that reduced ALK1 activity reduces survival and promotes cardiac fibrosis after MI. MI was induced in wild-type (WT), and ALK+/- mice by left coronary ligation. After 14 days ALK1+/- mice had reduced survival with a higher rate of cardiac rupture compared to WT mice. ALK1+/- left ventricles (LVs) had increased volumes at the end of systole and at the end of diastole. After MI ALK1+/- LVs had increased profibrotic SMAD3 signaling, type 1 collagen, and fibrosis as well as increased levels of TGFß1 co-receptor, endoglin, VEGF, and ALK1 ligands BMP9 and BMP10. ALK1+/- LVs had decreased levels of stromal-derived factor 1α. These data identify the critical role of ALK1 in post-MI survival and cardiac remodeling and implicate ALK1 as a potential therapeutic target to improve survival after MI.

Loss of Bone Morphogenetic Protein-9 Reduces Survival and Increases MMP Activity After Myocardial Infarction.

No studies have explored a functional role for bone morphogenetic protein (BMP)-9, a transforming growth factor-ß superfamily ligand, in cardiac remodeling after myocardial infarction (MI). Using BMP-9 null mice, we observed that loss of BMP-9 decreases survival and increases cardiac rupture after MI. We further observed that loss of BMP-9 not only increases collagen abundance, but also promotes matrix metalloproteinase-9 activity and collagen degradation after MI. These findings identify BMP-9 as a necessary component of cardiac remodeling after MI and a potentially important target of therapy to improve outcomes after MI.

Transvalvular Unloading Mitigates Ventricular Injury Due to Venoarterial Extracorporeal Membrane Oxygenation in Acute Myocardial Infarction.

Whether extracorporeal membrane oxygenation (ECMO) with Impella, known as EC-Pella, limits cardiac damage in acute myocardial infarction remains unknown. The authors now report that the combination of transvalvular unloading and ECMO (EC-Pella) initiated before reperfusion reduced infarct size compared with ECMO alone before reperfusion in a preclinical model of acute myocardial infarction. EC-Pella also reduced left ventricular pressure-volume area when transvalvular unloading was applied before, not after, activation of ECMO. The authors further observed that EC-Pella increased cardioprotective signaling but failed to rescue mitochondrial dysfunction compared with ECMO alone. These findings suggest that ECMO can increase infarct size in acute myocardial infarction and that EC-Pella can mitigate this effect but also suggest that left ventricular unloading and myocardial salvage may be uncoupled in the presence of ECMO in acute myocardial infarction. These observations implicate mechanisms beyond hemodynamic load as part of the injury cascade associated with ECMO in acute myocardial infarction.

Myocardial Injury Promotes Matrix Metalloproteinase-9 Activity in the Renal Cortex in Preclinical Models of Acute Myocardial Infarction.

New mechanistic insight into how the kidney responds to cardiac injury during acute myocardial infarction (AMI) is required. We hypothesized that AMI promotes inflammation and matrix metalloproteinase-9 (MMP9) activity in the kidney and studied the effect of initiating an Impella CP or veno-arterial extracorporeal membrane oxygenation (VA-ECMO) before coronary reperfusion during AMI. Adult male swine were subjected to coronary occlusion and either reperfusion (ischemia-reperfusion; IR) or support with either Impella or VA-ECMO before reperfusion. IR and ECMO increased while Impella reduced levels of MMP-9 in the myocardial infarct zone, circulation, and renal cortex. Compared to IR, Impella reduced myocardial infarct size and urinary KIM-1 levels, but VA-ECMO did not. IR and VA-ECMO increased pro-fibrogenic signaling via transforming growth factor-beta and endoglin in the renal cortex, but Impella did not. These findings identify that AMI increases inflammatory activity in the kidney, which may be attenuated by Impella support.

Long-term outcomes of infantile spasms in children treated with ketogenic diet therapy in combination with anti-seizure medications in a resource-limited region.

Objective: Despite numerous guidelines, the overall outcome of infantile spasms is poor, with only a small number of patients being able to attend school. The purpose of this study was to investigate long-term outcomes. Patients had poor access to the recommended first-line anti-seizure medications (ASMs), such as hormones (corticotropin or prednisolone/prednisone) and vigabatrin, and their alternative treatment was other ASMs and a ketogenic diet. Methods: Patients suffering from infantile spasms who had at least 2 years of medical records in the electronic medical record system between January 2014 and August 2022 were included in this study. Patient information was retrospectively reviewed. All patients had received ketogenic diet therapy (mainly classical ketogenic diet therapy). The ketogenic diet therapy was combined with ASMs not used as first-line therapies. The primary endpoint outcome measure was the number of patients with seizure freedom. The secondary measures included the duration of ketogenic diet therapy, choice of ASMs, and patient development at the last visit. Results: A total of 177 patients with infantile spasms were included, and 152 (86%) of them had seizure freedom. The median duration from the first to the last hospital visit was 53.27 months, and the number of visits was 47.00. The median age at the initial hospital visit was 8.00 months, and the median age at initiation of the ketogenic diet was 17.73 months. At the last visit, the proportions of patients with neurodevelopmental delay, developmental epileptic encephalopathy, drug-resistant epilepsy, and generalized seizures increased significantly. The frequently used ASMs were topiramate, valproic acid, levetiracetam, nitrazepam, and vitamin B6 injection, while the recommended first-line drugs corticotropin and vigabatrin were rarely selected. The study duration of 9.5 years was divided into three periods but the prescription of ASMs did not change significantly between these periods. Conclusions: Although the seizure freedom rate was high with ketogenic diet therapy combined with non-standard ASMs, the patients had a significant neurodevelopmental delay at the last visit, which was, however, similar to that of standard treatment. To improve the outcomes of infantile spasms, multicenter clinical trials of the ketogenic diet as a first-line treatment in combination with non-standard ASMs are needed.

Ran overexpression leads to diminished T cell responses and selectively modulates nuclear levels of c-Jun and c-Fos.

Ras-related nuclear protein (Ran) is a Ras family GTPase, and its documented functions are the regulation of DNA replication, cell cycle progression, nuclear structure formation, RNA processing and exportation, and nuclear protein importation. In this study, we performed detailed mapping of Ran expression during mouse ontogeny using in situ hybridization. High Ran expression was found in various organs and tissues including the thymus cortex and spleen white pulp. Ran was induced in T cells 24 h after their activation. The function of Ran in the immune system was investigated using Ran transgenic (Tg) mice. In Ran Tg T cells, there was compromised activation marker expression, lymphokine secretion, and proliferation upon T cell receptor activation in vitro when compared with wild type T cells. Tg mice also manifested defective delayed type hypersensitivity in vivo. Upon PMA and ionomycin stimulation, Tg T cells were defective in nuclear accumulation of AP-1 factors (c-Jun and c-Fos) but not NF-kappaB family members. Our experiments showed that Ran had important regulatory function in T cell activation. One of the possible mechanisms is that intracellular Ran protein levels control the nuclear retention for selective transcription factors such as c-Jun and c-Fos of AP-1, which is known to be critical in T cell activation and proliferation and lymphokine secretion.

Prolonged mechanical stretch is associated with upregulation of hypoxia-inducible factors and reduced contraction in rat inferior vena cava.

BACKGROUND: Decreased venous tone and vein wall dilation may contribute to varicose vein formation. We have shown that prolonged vein wall stretch is associated with upregulation of matrix metalloproteases (MMPs) and decreased contraction. Because hypoxia-inducible factors (HIFs) expression also increases with mechanical stretch, this study tested whether upregulation of HIFs is an intermediary mechanism linking prolonged vein wall stretch to the changes in MMP expression and venous contraction. METHODS: Segments of rat inferior vena cava (IVC) were suspended in tissue bath under 0.5-g basal tension for 1 hour, and a control contraction to phenylephrine (PHE, 10(-5)M) and KCl (96 mM) was elicited. The veins were then exposed to prolonged 18 hours of tension at 0.5 g, 2 g, 2 g plus HIF inhibitor U0126 (10(-5)M), 17-[2-(dimethylamino)ethyl] amino-17-desmethoxygeldanamycin (17-DMAG, 10(-5)M), or echinomycin (10(-6)M), or 2 g plus dimethyloxallyl glycine (DMOG; 10(-4)M), a prolyl-hydroxylase inhibitor that stabilizes HIF. The fold-change in PHE and KCl contraction was compared with the control contraction at 0.5-g tension for 1 hour. Vein tissue homogenates were analyzed for HIF-1α, HIF-2α, MMP-2, and MMP-9 messenger RNA (mRNA) and protein amount using real-time reverse transcription polymerase chain reaction and Western blots. RESULTS: Compared with control IVC contraction at 0.5-g tension for 1 hour, the PHE and KCl contraction after prolonged 0.5-g tension was 2.0 ± 0.35 and 1.1 ± 0.06, respectively. Vein contraction to PHE and KCl after prolonged 2-g tension was significantly reduced (0.87 ± 0.13 and 0.72 ± 0.05, respectively). PHE-induced contraction was restored in IVC exposed to prolonged 2-g tension plus the HIF inhibitor U0126 (1.38 ± 0.15) or echinomycin (1.99 ± 0.40). U0126 and echinomycin also restored KCl-induced contraction in IVC exposed to prolonged 2-g tension (1.14 ± 0.05 and 1.11 ± 0.15, respectively). Treatment with DMOG further reduced PHE- and KCl-induced contraction in veins subjected to prolonged 2-g tension (0.47 ± 0.06 and 0.57 ± 0.01, respectively). HIF-1α and HIF-2α mRNA were overexpressed in IVC exposed to prolonged 2-g tension, and the overexpression was reversed by U0126. The overexpression of HIF-1α and HIF-2α in stretched IVC was associated with increased MMP-2 and MMP-9 mRNA. The protein amount of HIF-1α, HIF-2α, MMP-2, and MMP-9 was also increased in IVC exposed to prolonged 2-g wall tension. CONCLUSIONS: Prolonged increases in vein wall tension are associated with overexpression of HIF-1α and HIF-2α, increased MMP-2 and MMP-9 expression, and reduced venous contraction in rat IVC. Together with our report that MMP-2 and MMP-9 inhibit IVC contraction, the data suggest that increased vein wall tension induces HIF overexpression and causes an increase in MMP expression and reduction of venous contraction, leading to progressive venous dilation and varicose vein formation.

Gender-specific reduction in contraction is associated with increased estrogen receptor expression in single vascular smooth muscle cells of female rat.

Gender differences in the incidence of cardiovascular disease have been related to plasma estrogen levels; however, the role of vascular estrogen receptor (ER) subtypes in these sex differences is less clear. We tested whether the gender differences in vascular smooth muscle (VSM) function reflect differential expression/activity of ERalpha, ERbeta and the newly-identified GPR30. Single aortic VSM cells (VSMCs) were freshly isolated from male and female Sprague-Dawley rats, and their contraction to phenylephrine (PHE, 10(-5) M), AngII (10(-7) M) and membrane-depolarization by KCl (51 mM) was measured in the absence or presence of 10(-6) M 17beta-estradiol (E2, stimulant of most ERs), PPT (ERalpha agonist), DPN (ERbeta agonist), and ICI 182,780 (an ERalpha/ERbeta antagonist with GPR30 agonistic properties). The cells were fixed and fluorescently labeled with ERalpha, ERbeta or GPR30 antibody, and the subcellular distribution of ERs was examined using digital imaging microscopy. The mRNA expression and protein amount of aortic ER subtypes was examined using RT-PCR and Western blots. PHE, AngII, and KCl caused less contraction in VSMCs of females than males. Pretreatment of VSMCs with E2 reduced PHE-, AngII- and KCl-induced contraction in both males and females. PPT caused similar inhibition of PHE-, AngII- and KCl-induced contraction as E2, suggesting a role of ERalpha. DPN mainly inhibited PHE and KCl contraction, suggesting an interaction between ERbeta and Ca(2+) channels. ICI 182,780 did not reduce aortic VSMC contraction, suggesting little role for GPR30. RT-PCR and Western blots revealed greater expression of ERalpha and ERbeta in VSMCs of females than males, but similar amounts of GPR30. The total immunofluorescence signal for ERalpha and ERbeta was greater in VSMCs of females than males, and was largely localized in the nucleus. GPR30 fluorescence was similar in VSMCs of males and females, and was mainly in the cytosol. In PPT treated cells, nuclear ERalpha signal was enhanced. DPN did not affect the distribution of ERbeta, and ICI 182,780 did not significantly increase GPR30 in the cell surface. Thus, ER subtypes demonstrate similar responsiveness to specific agonists in VSMCs of male and female rats. The reduced contraction in VSMCs of females could be due to gender-related increase in the expression of ERalpha and ERbeta.

Estrogen receptor-mediated enhancement of venous relaxation in female rat: implications in sex-related differences in varicose veins.

BACKGROUND: A greater incidence of varicose veins has been reported in premenopausal women than in men. We hypothesized that the sex differences in venous function reflect reduced constriction and enhanced venous dilation in women due to direct venous relaxation effects of estrogen on specific estrogen receptors (ER). METHODS: Circular segments of inferior vena cava (IVC) from male and female Sprague-Dawley rats were suspended between two wires, and isometric contraction (in mg/mg tissue) to phenylephrine, angiotensin II (AngII), and 96 mM KCl was measured. To investigate sex differences in venous smooth muscle, Ca(2+) release from the intracellular stores, and Ca(2+) entry from the extracellular space, the transient phenylephrine contraction in 0 Ca(2+) Krebs was measured. Extracellular CaCl(2) (0.1, 0.3, 0.6, 1, 2.5 mM) was added, and the [Ca(2+)](e)-dependent contraction was measured. To investigate sex differences in venous endothelial function, acetylcholine-induced relaxation was measured. To test the role of specific ERs, the amount of venous tissue ERs was measured using Western blots, and the venous relaxation in response to 17beta-estradiol (E2, activator of most ERs), 4,4,'4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)-tris-phenol (PPT; ERalpha agonist), 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; ERbeta agonist), and ICI 182,780 (ERalpha/ERbeta antagonist, and G protein-coupled receptor 30 [GPR30] agonist) was measured in IVC segments nontreated or treated with the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). RESULTS: Phenylephrine caused concentration-dependent contraction that was less in female (max 104.2 +/- 16.2) than male IVC (172.4 +/- 20.4). AngII (10(-6))-induced contraction was also less in female (81.0 +/- 11.1) than male IVC (122.5 +/- 15.0). Phenylephrine contraction in 0 Ca(2+) Krebs was insignificantly less in female (4.8 +/- 1.8) than male IVC (7.2 +/- 1.7), suggesting little difference in the intracellular Ca(2+) release mechanism. In contrast, the [Ca(2+)](e)-dependent contraction was significantly reduced in female than male IVC. Also, contraction to membrane depolarization by 96 mM KCl, which stimulates Ca(2+) influx, was less in female (129.7 +/- 16.7) than male IVC (319.7 +/- 30.4), supporting sex differences in Ca(2+) entry. Acetylcholine relaxation was greater in female (max 80.6% +/- 4.1%) than male IVC (max 48.0% +/- 6.1%), suggesting sex differences in the endothelium-dependent relaxation pathway. Western blots revealed greater amounts of ERalpha, ERbeta, and GPR30 in female than male IVC. ER agonists caused concentration-dependent relaxation of phenylephrine contraction in female IVC. E2-induced relaxation (max 76.5% +/- 3.4%) was more than DPN (74.8% +/- 9.1%), PPT (71.4% +/- 12.5%), and ICI 182,780 (67.4% +/- 7.8%), and was similar in L-NAME-treated and nontreated IVC. CONCLUSION: The reduced alpha-adrenergic, AngII, depolarization-induced, and [Ca(2+)](e)-dependent venous contraction in female rats is consistent with sex differences in the Ca(2+) entry mechanisms, possibly due to enhanced endothelium-dependent vasodilation and increased ER expression/activity in female rats. E2/ER-mediated venous relaxation in female rats is not prevented by NOS blockade, suggesting activation of an NO-independent relaxation pathway. The decreased venous contraction and enhanced E2/ER-mediated venous relaxation would lead to more distensible veins in female rats.