The Loss of an Orphan Nuclear Receptor NR2E3 Augments Wnt/ß-catenin Signaling via Epigenetic Dysregulation that Enhances Sp1-ß catenin-p300 Interactions in Hepatocellular Carcinoma.

The orphan nuclear receptor NR2E3 (Nuclear receptor subfamily 2 group E, Member 3) is an epigenetic player that modulates chromatin accessibility to activate p53 during liver injury. Nonetheless, a precise tumor suppressive and epigenetic role of NR2E3 in hepatocellular carcinoma (HCC) development remains unclear. HCC patients expressing low NR2E3 exhibit unfavorable clinical outcomes, aligning with heightened activation of the Wnt/ß-catenin signaling pathway. The murine HCC models utilizing NR2E3 knockout mice consistently exhibits accelerated liver tumor formation accompanied by enhanced activation of Wnt/ß-catenin signaling pathway and inactivation of p53 signaling. At cellular level, the loss of NR2E3 increases the acquisition of aggressive cancer cell phenotype and tumorigenicity and upregulates key genes in the WNT/ß-catenin pathway with increased chromatin accessibility. This event is mediated through increased formation of active transcription complex involving Sp1, ß-catenin, and p300, a histone acetyltransferase, on the promoters of target genes. These findings demonstrate that the loss of NR2E3 activates Wnt/ß-catenin signaling at cellular and organism levels and this dysregulation is associated with aggressive HCC development and poor clinical outcomes. In summary, NR2E3 is a novel tumor suppressor with a significant prognostic value, maintaining epigenetic homeostasis to suppress the Wnt/ß-catenin signaling pathway that promotes HCC development.

Role of myeloid cells in ischemic retinopathies: recent advances and unanswered questions.

Myeloid cells including microglia and macrophages play crucial roles in retinal homeostasis by clearing cellular debris and regulating inflammation. These cells are activated in several blinding ischemic retinal diseases including diabetic retinopathy, where they may exert both beneficial and detrimental effects on neurovascular function and angiogenesis. Myeloid cells impact the progression of retinal pathologies and recent studies suggest that targeting myeloid cells is a promising therapeutic strategy to mitigate diabetic retinopathy and other ischemic retinal diseases. This review summarizes the recent advances in our understanding of the role of microglia and macrophages in retinal diseases and focuses on the effects of myeloid cells on neurovascular injury and angiogenesis in ischemic retinopathies. We highlight gaps in knowledge and advocate for a more detailed understanding of the role of myeloid cells in retinal ischemic injury to fully unlock the potential of targeting myeloid cells as a therapeutic strategy for retinal ischemia.

The arginase 1/ornithine decarboxylase pathway suppresses HDAC3 to ameliorate the myeloid cell inflammatory response: implications for retinal ischemic injury.

The enzyme arginase 1 (A1) hydrolyzes the amino acid arginine to form L-ornithine and urea. Ornithine is further converted to polyamines by the ornithine decarboxylase (ODC) enzyme. We previously reported that deletion of myeloid A1 in mice exacerbates retinal damage after ischemia/reperfusion (IR) injury. Furthermore, treatment with A1 protects against retinal IR injury in wild-type mice. PEG-A1 also mitigates the exaggerated inflammatory response of A1 knockout (KO) macrophages in vitro. Here, we sought to identify the anti-inflammatory pathway that confers macrophage A1-mediated protection against retinal IR injury. Acute elevation of intraocular pressure was used to induce retinal IR injury in mice. A multiplex cytokine assay revealed a marked increase in the inflammatory cytokines interleukin 1ß (IL-1ß) and tumor necrosis factor α (TNF-α) in the retina at day 5 after IR injury. In vitro, blocking the A1/ODC pathway augmented IL-1ß and TNF-α production in stimulated macrophages. Furthermore, A1 treatment attenuated the stimulated macrophage metabolic switch to a pro-inflammatory glycolytic phenotype, whereas A1 deletion had the opposite effect. Screening for histone deacetylases (HDACs) which play a role in macrophage inflammatory response showed that A1 deletion or ODC inhibition increased the expression of HDAC3. We further showed the involvement of HDAC3 in the upregulation of TNF-α but not IL-1ß in stimulated macrophages deficient in the A1/ODC pathway. Investigating HDAC3 KO macrophages showed a reduced inflammatory response and a less glycolytic phenotype upon stimulation. In vivo, HDAC3 co-localized with microglia/macrophages at day 2 after IR in WT retinas and was further increased in A1-deficient retinas. Collectively, our data provide initial evidence that A1 exerts its anti-inflammatory effect in macrophages via ODC-mediated suppression of HDAC3 and IL-1ß. Collectively we propose that interventions that augment the A1/ODC pathway and inhibit HDAC3 may confer therapeutic benefits for the treatment of retinal ischemic diseases.

PCSK9 attenuates efferocytosis in endothelial cells and promotes vascular aging.

Aims: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that binds to low-density lipoprotein receptors. Efferocytosis is the process by which phagocytes remove apoptotic cells. Both PCSK9 and efferocytosis play important roles in regulating redox biology and inflammation, the key factors contributing to vascular aging. This study was designed to investigate the impact of PCSK9 on efferocytosis in endothelial cells (ECs) and its implications in vascular aging. Methods and Results: Studies were performed in primary human aortic ECs (HAECs) and primary mouse aortic ECs (MAECs) isolated from male wild-type (WT) and PCSK9-/- mice, and in young and aged mice treated with saline or the PCSK9 inhibitor Pep2-8. Our findings include that recombinant PCSK9 protein induces defective efferocytosis and aging marker senescence-associated-ß-galactosidase (SA-ß-gal) expression in ECs, while PCSK9-/- restores efferocytosis and inhibits SA-ß-gal activity. Further studies in aged mice showed that endothelial deficiency of MerTK, a critical receptor for efferocytosis that allows phagocytes to detect the presence of apoptotic cells, may be an indicator of vascular dysfunction in the aortic arch. Pep2-8 treatment markedly restored efferocytosis in endothelium from the aged mice. A proteomics study in the aortic arch from aged mice revealed that Pep2-8 administration significantly downregulates expression of NOX4, MAPK subunits, NF-κB, and secretion of pro-inflammatory cytokines, all known to promote vascular aging. Immunofluorescent staining showed that Pep2-8 administration upregulates expression of eNOS and downregulates expression of pro-IL-1ß, NF-κB and p22phox compared to saline treated group. Conclusions: These findings provide initial evidence for the ability of aortic ECs to accomplish efferocytosis and argue for a role of PCSK9 in attenuating EC efferocytosis, thereby leading to vascular dysfunction and acceleration in vascular aging.

Mitochondrial proton leak in cardiac aging.

Age-associated diseases are becoming progressively more prevalent, reflecting the increased lifespan of the world's population. However, the fundamental mechanisms of physiologic aging are poorly understood, and in particular, the molecular pathways that mediate cardiac aging and its associated dysfunction are unclear. Here, we focus on certain ion flux abnormalities of the mitochondria that may contribute to cardiac aging and age-related heart failure. Using oxidative phosphorylation, mitochondria pump protons from the matrix to the intermembrane space to generate a proton gradient across the inner membrane. The protons are returned to the matrix by the ATPase complex within the membrane to generate ATP. However, a portion of protons leak back to the matrix and do not drive ATP production, and this event is called proton leak or uncoupling. Accumulating evidence suggests that mitochondrial proton leak is increased in the cardiac myocytes of aged hearts. In this mini-review, we discuss the measurement methods and major sites of mitochondrial proton leak with an emphasis on the adenine nucleotide transporter 1 (ANT1), and explore the possibility of inhibiting augmented mitochondrial proton leak as a therapeutic intervention to mitigate cardiac aging.

Drug-Related Lymphedema: Mysteries, Mechanisms, and Potential Therapies.

The lymphatic circulation is an important component of the circulatory system in humans, playing a critical role in the transport of lymph fluid containing proteins, white blood cells, and lipids from the interstitial space to the central venous circulation. The efficient transport of lymph fluid critically relies on the rhythmic contractions of collecting lymph vessels, which function to "pump" fluid in the distal to proximal direction through the lymphatic circulation with backflow prevented by the presence of valves. When rhythmic contractions are disrupted or valves are incompetent, the loss of lymph flow results in fluid accumulation in the interstitial space and the development of lymphedema. There is growing recognition that many pharmacological agents modify the activity of ion channels and other protein structures in lymph muscle cells to disrupt the cyclic contraction and relaxation of lymph vessels, thereby compromising lymph flow and predisposing to the development of lymphedema. The effects of different medications on lymph flow can be understood by appreciating the intricate intracellular calcium signaling that underlies the contraction and relaxation cycle of collecting lymph vessels. For example, voltage-sensitive calcium influx through long-lasting ("L-type") calcium channels mediates the rise in cytosolic calcium concentration that triggers lymph vessel contraction. Accordingly, calcium channel antagonists that are mainstay cardiovascular medications, attenuate the cyclic influx of calcium through L-type calcium channels in lymph muscle cells, thereby disrupting rhythmic contractions and compromising lymph flow. Many other classes of medications also may contribute to the formation of lymphedema by impairing lymph flow as an off-target effect. The purpose of this review is to evaluate the evidence regarding potential mechanisms of drug-related lymphedema with an emphasis on common medications administered to treat cardiovascular diseases, metabolic disorders, and cancer. Additionally, although current pharmacological approaches used to alleviate lymphedema are largely ineffective, efforts are mounting to arrive at a deeper understanding of mechanisms that regulate lymph flow as a strategy to identify novel anti-lymphedema medications. Accordingly, this review also will provide information on studies that have explored possible anti-lymphedema therapeutics.

Dantrolene Prevents the Lymphostasis Caused by Doxorubicin in the Rat Mesenteric Circulation.

Background and Purpose: Doxorubicin (DOX) is a risk factor for arm lymphedema in breast cancer patients. We reported that DOX opens ryanodine receptors (RYRs) to enact "calcium leak," which disrupts the rhythmic contractions of lymph vessels (LVs) to attenuate lymph flow. Here, we evaluated whether dantrolene, a clinically available RYR1 subtype antagonist, prevents the detrimental effects of DOX on lymphatic function. Experimental Approach: Isolated rat mesenteric LVs were cannulated, pressurized (4-5 mm Hg) and equilibrated in physiological salt solution and Fura-2AM. Video microscopy recorded changes in diameter and Fura-2AM fluorescence tracked cytosolic free calcium ([Ca2+ i]). High-speed in vivo microscopy assessed mesenteric lymph flow in anesthetized rats. Flow cytometry evaluated RYR1 expression in freshly isolated mesenteric lymphatic muscle cells (LMCs). Key Results: DOX (10 µmol/L) increased resting [Ca2+ i] by 17.5 ± 3.7% in isolated LVs (n = 11). The rise in [Ca2+ i] was prevented by dantrolene (3 µmol/L; n = 10). A single rapid infusion of DOX (10 mg/kg i.v.) reduced positive volumetric lymph flow to 29.7 ± 10.8% (n = 7) of baseline in mesenteric LVs in vivo. In contrast, flow in LVs superfused with dantrolene (10 µmol/L) only decreased to 76.3 ± 14.0% (n = 7) of baseline in response to DOX infusion. Subsequently, expression of the RYR1 subtype protein as the presumed dantrolene binding site was confirm in isolated mesenteric LMCs by flow cytometry. Conclusion and Implications: We conclude that dantrolene attenuates the acute impairment of lymph flow by DOX and suggest that its prophylactic use in patients subjected to DOX chemotherapy may lower lymphedema risk.

KATP Channel Openers Inhibit Lymphatic Contractions and Lymph Flow as a Possible Mechanism of Peripheral Edema.

Pharmacological openers of ATP-sensitive potassium (KATP) channels are effective antihypertensive agents, but off-target effects, including severe peripheral edema, limit their clinical usefulness. It is presumed that the arterial dilation induced by KATP channel openers (KCOs) increases capillary pressure to promote filtration edema. However, KATP channels also are expressed by lymphatic muscle cells (LMCs), raising the possibility that KCOs also attenuate lymph flow to increase interstitial fluid. The present study explored the effect of KCOs on lymphatic contractile function and lymph flow. In isolated rat mesenteric lymph vessels (LVs), the prototypic KATP channel opener cromakalim (0.01-3 µmol/l) progressively inhibited rhythmic contractions and calculated intraluminal flow. Minoxidil sulfate and diazoxide (0.01-100 µmol/l) had similar effects at clinically relevant plasma concentrations. High-speed in vivo imaging of the rat mesenteric lymphatic circulation revealed that superfusion of LVs with cromakalim and minoxidil sulfate (0.01-10 µmol/l) maximally decreased lymph flow in vivo by 38.4% and 27.4%, respectively. Real-time polymerase chain reaction and flow cytometry identified the abundant KATP channel subunits in LMCs as the pore-forming Kir6.1/6.2 and regulatory sulfonylurea receptor 2 subunits. Patch-clamp studies detected cromakalim-elicited unitary K+ currents in cell-attached patches of LMCs with a single-channel conductance of 46.4 pS, which is a property consistent with Kir6.1/6.2 tetrameric channels. Addition of minoxidil sulfate and diazoxide elicited unitary currents of similar amplitude. Collectively, our findings indicate that KCOs attenuate lymph flow at clinically relevant plasma concentrations as a potential contributing mechanism to peripheral edema. SIGNIFICANCE STATEMENT: ATP-sensitive potassium (KATP) channel openers (KCOs) are potent antihypertensive medications, but off-target effects, including severe peripheral edema, limit their clinical use. Here, we demonstrate that KCOs impair the rhythmic contractions of lymph vessels and attenuate lymph flow, which may promote edema formation. Our finding that the KATP channels in lymphatic muscle cells may be unique from their counterparts in arterial muscle implies that designing arterial-selective KCOs may avoid activation of lymphatic KATP channels and peripheral edema.

Metoprolol Impairs ß1-Adrenergic Receptor-Mediated Vasodilation in Rat Cerebral Arteries: Implications for ß-Blocker Therapy.

The practice of prescribing ß-blockers to lower blood pressure and mitigate perioperative cardiovascular events has been questioned because of reports of an increased risk of stroke. The benefit of ß-blocker therapy primarily relies on preventing activation of cardiac ß1-adrenergic receptors (ARs). However, we reported that ß1ARs also mediate vasodilator responses of rat cerebral arteries (CAs), implying that ß-blockers may impair cerebral blood flow under some conditions. Here, we defined the impact of metoprolol (MET), a widely prescribed ß1AR-selective antagonist, on adrenergic-elicited diameter responses of rat CAs ex vivo and in vivo. MET (1-10 µmol/l) prevented ß1AR-mediated increases in diameter elicited by dobutamine in cannulated rat CAs. The ß1AR-mediated dilation elicited by the endogenous adrenergic agonist norepinephrine (NE) was reversed to a sustained constriction by MET. Acute oral administration of MET (30 mg/kg) to rats in doses that attenuated resting heart rate and dobutamine-induced tachycardia also blunted ß1AR-mediated dilation of CAs. In the same animals, NE-induced dilation of CAs was reversed to sustained constriction. Administration of MET for 2 weeks in drinking water (2 mg/ml) or subcutaneously (15 mg/kg per day) also resulted in NE-induced constriction of CAs in vivo. Thus, doses of MET that protect the heart from adrenergic stimulation also prevent ß1AR-mediated dilation of CAs and favor anomalous adrenergic constriction. Our findings raise the possibility that the increased risk of ischemic stroke in patients on ß-blockers relates in part to adrenergic dysregulation of cerebrovascular tone. SIGNIFICANCE STATEMENT: ß-Blocker therapy using second-generation, cardioselective ß-blockers is associated with an increased risk of stroke, but the responsible mechanisms are unclear. Here, we report that either acute or chronic systemic administration of a cardioselective ß-blocker, metoprolol, mitigates adrenergic stimulation of the heart as an intended beneficial action. However, metoprolol concomitantly eliminates vasodilator responses to adrenergic stimuli of rat cerebral arteries in vivo as a potential cause of dysregulated cerebral blood flow predisposing to ischemic stroke.

Vascular smooth muscle TRPC3 channels facilitate the inverse hemodynamic response during status epilepticus.

Human status epilepticus (SE) is associated with a pathological reduction in cerebral blood flow termed the inverse hemodynamic response (IHR). Canonical transient receptor potential 3 (TRPC3) channels are integral to the propagation of seizures in SE, and vascular smooth muscle cell (VSMC) TRPC3 channels participate in vasoconstriction. Therefore, we hypothesize that cerebrovascular TRPC3 channels may contribute to seizure-induced IHR. To examine this possibility, we developed a smooth muscle-specific TRPC3 knockout (TRPC3smcKO) mouse. To quantify changes in neurovascular coupling, we combined laser speckle contrast imaging with simultaneous electroencephalogram recordings. Control mice exhibited multiple IHRs, and a limited increase in cerebral blood flow during SE with a high degree of moment-to-moment variability in which blood flow was not correlated with neuronal activity. In contrast, TRPC3smcKO mice showed a greater increase in blood flow that was less variable and was positively correlated with neuronal activity. Genetic ablation of smooth muscle TRPC3 channels shortened the duration of SE by eliminating a secondary phase of intense seizures, which was evident in littermate controls. Our results are consistent with the idea that TRPC3 channels expressed by cerebral VSMCs contribute to the IHR during SE, which is a critical factor in the progression of SE.

Specific BK Channel Activator NS11021 Protects Rat Renal Proximal Tubular Cells from Cold Storage-Induced Mitochondrial Injury In Vitro.

Kidneys from deceased donors used for transplantation are placed in cold storage (CS) solution during the search for a matched recipient. However, CS causes mitochondrial injury, which may exacerbate renal graft dysfunction. Here, we explored whether adding NS11021, an activator of the mitochondrial big-conductance calcium-activated K+ (mitoBK) channel, to CS solution can mitigate CS-induced mitochondrial injury. We used normal rat kidney proximal tubular epithelial (NRK) cells as an in vitro model of renal cold storage (18 h) and rewarming (2 h) (CS + RW). Western blots detected the pore-forming α subunit of the BK channel in mitochondrial fractions from NRK cells. The fluorescent K+-binding probe, PBFI-AM, revealed that isolated mitochondria from NRK cells exhibited mitoBK-mediated K+ uptake, which was impaired ~70% in NRK cells subjected to CS + RW compared to control NRK cells maintained at 37 °C. Importantly, the addition of 1 M NS11021 to CS solution prevented CS + RW-induced impairment of mitoBK-mediated K+ uptake. The NS11021-treated NRK cells also exhibited less cell death and mitochondrial injury after CS + RW, including mitigated mitochondrial respiratory dysfunction, depolarization, and superoxide production. In summary, these new data show for the first time that mitoBK channels may represent a therapeutic target to prevent renal CS-induced injury.

Doxorubicin Activates Ryanodine Receptors in Rat Lymphatic Muscle Cells to Attenuate Rhythmic Contractions and Lymph Flow.

Doxorubicin is a risk factor for secondary lymphedema in cancer patients exposed to surgery or radiation. The risk is presumed to relate to its cytotoxicity. However, the present study provides initial evidence that doxorubicin directly inhibits lymph flow and this action appears distinct from its cytotoxic activity. We used real-time edge detection to track diameter changes in isolated rat mesenteric lymph vessels. Doxorubicin (0.5-20 µmol/l) progressively constricted lymph vessels and inhibited rhythmic contractions, reducing flow to 24.2% ± 7.7% of baseline. The inhibition of rhythmic contractions by doxorubicin paralleled a tonic rise in cytosolic Ca2+ concentration in lymphatic muscle cells, which was prevented by pharmacological antagonism of ryanodine receptors. Washout of doxorubicin partially restored lymph vessel contractions, implying a pharmacological effect. Subsequently, high-speed optical imaging was used to assess the effect of doxorubicin on rat mesenteric lymph flow in vivo. Superfusion of doxorubicin (0.05-10 µmol/l) maximally reduced volumetric lymph flow to 34% ± 11.6% of baseline. Likewise, doxorubicin (10 mg/kg) administered intravenously to establish clinically achievable plasma concentrations also maximally reduced volumetric lymph flow to 40.3% ± 6.0% of initial values. Our findings reveal that doxorubicin at plasma concentrations achieved during chemotherapy opens ryanodine receptors to induce "calcium leak" from the sarcoplasmic reticulum in lymphatic muscle cells and reduces lymph flow, an event linked to lymph vessel damage and the development of lymphedema. These results infer that pharmacological block of ryanodine receptors in lymphatic smooth muscle cells may mitigate secondary lymphedema in cancer patients subjected to doxorubicin chemotherapy. SIGNIFICANCE STATEMENT: Doxorubicin directly inhibits the rhythmic contractions of collecting lymph vessels and reduces lymph flow as a possible mechanism of secondary lymphedema, which is associated with the administration of anthracycline-based chemotherapy. The inhibitory effects of doxorubicin on rhythmic contractions and flow in isolated lymph vessels were prevented by pharmacological block of ryanodine receptors, thereby identifying the ryanodine receptor family of proteins as potential therapeutic targets for the development of new antilymphedema medications.

High-speed microscopy for in vivo monitoring of lymph dynamics.

The lymphatic system contributes to body homeostasis by clearing fluid, lipids, plasma proteins and immune cells from the interstitial space. Many studies have been performed to understand lymphatic function under normal conditions and during disease. Nevertheless, a further improvement in quantification of lymphatic behavior is needed. Here, we present advanced bright-field microscopy for in vivo imaging of lymph vessels (LVs) and automated quantification of lymphatic function at a temporal resolution of 2 milliseconds. Full frame videos were compressed and recorded continuously at up to 540 frames per second. A new edge detection algorithm was used to monitor vessel diameter changes across multiple cross sections, while individual cells in the LVs were tracked to estimate flow velocity. The system performance initially was verified in vitro using 6- and 10-µm microspheres as cell phantoms on slides and in 90-µm diameter tubes at flow velocities up to 4 cm/second. Using an in vivo rat model, we explored the mechanisms of lymphedema after surgical lymphadenectomy of the mesentery. The system revealed reductions of mesenteric LV contraction and flow rate. Thus, the described imaging system may be applicable to the study of lymphatic behavior during therapeutic and surgical interventions, and potentially during lymphatic system diseases.

Molecular determinants of beta-adrenergic signaling to voltage-gated K+ channels in the cerebral circulation.

Voltage-gated K+ (Kv ) channels are major determinants of membrane potential in vascular smooth muscle cells (VSMCs) and regulate the diameter of small cerebral arteries and arterioles. However, the intracellular structures that govern the expression and function of vascular Kv channels are poorly understood. Scaffolding proteins including postsynaptic density 95 (PSD95) recently were identified in rat cerebral VSMCs. Primarily characterized in neurons, the PSD95 scaffold has more than 50 known binding partners, and it can mediate macromolecular signaling between cell-surface receptors and ion channels. In cerebral arteries, Shaker-type Kv 1 channels appear to associate with the PSD95 molecular scaffold, and PSD95 is required for the normal expression and vasodilator influence of members of this K+ channel gene family. Furthermore, recent findings suggest that the ß1-subtype adrenergic receptor is expressed in cerebral VSMCs and forms a functional vasodilator complex with Kv 1 channels on the PSD95 scaffold. Activation of ß1-subtype adrenergic receptors in VSMCs enables protein kinase A-dependent phosphorylation and opening of Kv 1 channels in the PSD95 complex; the subsequent K+ efflux mediates membrane hyperpolarization and vasodilation of small cerebral arteries. Early evidence from other studies suggests that other families of Kv channels and scaffolding proteins are expressed in VSMCs. Future investigations into these macromolecular complexes that modulate the expression and function of Kv channels may reveal unknown signaling cascades that regulate VSMC excitability and provide novel targets for ion channel-based medications to optimize vascular tone.

17ß-estradiol reduces Cav 1.2 channel abundance and attenuates Ca2+ -dependent contractions in coronary arteries.

One mechanism by which the female sex may protect against elevated coronary vascular tone is inhibition of Ca2+ entry into arterial smooth muscle cells (ASMCs). In vitro findings confirm that high estrogen concentrations directly inhibit voltage-dependent Cav 1.2 channels in coronary ASMCs. For this study, we hypothesized that the nonacute, in vitro exposure of coronary arteries to a low concentration of 17ß-estradiol (17ßE) reduces the expression of Cav 1.2 channel proteins in coronary ASMCs. Segments of the right coronary artery obtained from sexually mature female pigs were mounted for isometric tension recording. As expected, our results indicate that high concentrations (≥10 µmol/L) of 17ßE acutely attenuated Ca2+ -dependent contractions to depolarizing KCl stimuli. Interestingly, culturing coronary arteries for 24 h in a 10,000-fold lower concentration (1 nmol/L) of 17ßE also attenuated KCl-induced contractions and reduced the contractile response to the Cav 1.2 agonist, FPL64176, by 50%. Western blots revealed that 1 nmol/L 17ßE decreased protein expression of the pore-forming α1C subunit (Cav α) of the Cav 1.2 channel by 35%; this response did not depend on an intact endothelium. The 17ßE-induced loss of Cav α protein in coronary arteries was prevented by the estrogen ERα/ERß antagonist, ICI 182,780, whereas the GPER antagonist, G15, did not prevent it. There was no effect of 1 nmol/L 17ßE on Cav α transcript expression. We conclude that 17ßE reduces Cav 1.2 channel abundance in isolated coronary arteries by a posttranscriptional process. This unrecognized effect of estrogen may confer physiological protection against the development of abnormal Ca2+ -dependent coronary vascular tone.

Thioredoxin reverses age-related hypertension by chronically improving vascular redox and restoring eNOS function.

The incidence of high blood pressure with advancing age is notably high, and it is an independent prognostic factor for the onset or progression of a variety of cardiovascular disorders. Although age-related hypertension is an established phenomenon, current treatments are only palliative but not curative. Thus, there is a critical need for a curative therapy against age-related hypertension, which could greatly decrease the incidence of cardiovascular disorders. We show that overexpression of human thioredoxin (TRX), a redox protein, in mice prevents age-related hypertension. Further, injection of recombinant human TRX (rhTRX) for three consecutive days reversed hypertension in aged wild-type mice, and this effect lasted for at least 20 days. Arteries of wild-type mice injected with rhTRX or mice with TRX overexpression exhibited decreased arterial stiffness, greater endothelium-dependent relaxation, increased nitric oxide production, and decreased superoxide anion (O2•-) generation compared to either saline-injected aged wild-type mice or mice with TRX deficiency. Our study demonstrates a potential translational role of rhTRX in reversing age-related hypertension with long-lasting efficacy.