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.

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.

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.

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.

Overexpression of the Large-Conductance, Ca2+-Activated K+ (BK) Channel Shortens Action Potential Duration in HL-1 Cardiomyocytes.

Long QT syndrome is characterized by a prolongation of the interval between the Q wave and the T wave on the electrocardiogram. This abnormality reflects a prolongation of the ventricular action potential caused by a number of genetic mutations or a variety of drugs. Since effective treatments are unavailable, we explored the possibility of using cardiac expression of the large-conductance, Ca2+-activated K+ (BK) channel to shorten action potential duration (APD). We hypothesized that expression of the pore-forming α subunit of human BK channels (hBKα) in HL-1 cells would shorten action potential duration in this mouse atrial cell line. Expression of hBKα had minimal effects on expression levels of other ion channels with the exception of a small but significant reduction in Kv11.1. Patch-clamped hBKα expressing HL-1 cells exhibited an outward voltage- and Ca2+-sensitive K+ current, which was inhibited by the BK channel blocker iberiotoxin (100 nM). This BK current phenotype was not detected in untransfected HL-1 cells or in HL-1 null cells sham-transfected with an empty vector. Importantly, APD in hBKα-expressing HL-1 cells averaged 14.3 ± 2.8 ms (n = 10), which represented a 53% reduction in APD compared to HL-1 null cells lacking BKα expression. APD in the latter cells averaged 31.0 ± 5.1 ms (n = 13). The shortened APD in hBKα-expressing cells was restored to normal duration by 100 nM iberiotoxin, suggesting that a repolarizing K+ current attributed to BK channels accounted for action potential shortening. These findings provide initial proof-of-concept that the introduction of hBKα channels into a cardiac cell line can shorten APD, and raise the possibility that gene-based interventions to increase hBKα channels in cardiac cells may hold promise as a therapeutic strategy for long QT syndrome.

Protein kinase A-phosphorylated KV1 channels in PSD95 signaling complex contribute to the resting membrane potential and diameter of cerebral arteries.

RATIONALE: Postsynaptic density-95 (PSD95) is a scaffolding protein that associates with voltage-gated, Shaker-type K(+) (KV1) channels and promotes the expression of KV1 channels in vascular smooth muscle cells of the cerebral (cVSMCs) circulation. However, the physiological role of PSD95 in mediating molecular signaling in cVSMCs is unknown. OBJECTIVE: We explored whether a specific interaction between PSD95 and KV1 channels enables protein kinase A phosphorylation of KV1 channels in cVSMCs to promote vasodilation. METHODS AND RESULTS: Rat cerebral arteries were used for analyses. A membrane-permeable peptide (KV1-C peptide) corresponding to the postsynaptic density-95, discs large, zonula occludens-1 binding motif in the C terminus of KV1.2α was designed as a dominant-negative peptide to disrupt the association of KV1 channels with PSD95. Application of KV1-C peptide to cannulated, pressurized cerebral arteries rapidly induced vasoconstriction and depolarized cVSMCs. These events corresponded to reduced coimmunoprecipitation of the PSD95 and KV1 proteins without altering surface expression. Middle cerebral arterioles imaged in situ through cranial window also constricted rapidly in response to local application of KV1-C peptide. Patch-clamp recordings confirmed that KV1-C peptide attenuates KV1 channel blocker (5-(4-phenylalkoxypsoralen))-sensitive current in cVSMCs. Western blots using a phospho-protein kinase A substrate antibody revealed that cerebral arteries exposed to KV1-C peptide showed markedly less phosphorylation of KV1.2α subunits. Finally, phosphatase inhibitors blunted both KV1-C peptide-mediated and protein kinase A inhibitor peptide-mediated vasoconstriction. CONCLUSIONS: These findings provide initial evidence that protein kinase A phosphorylation of KV1 channels is enabled by a dynamic association with PSD95 in cerebral arteries and suggest that a disruption of such association may compromise cerebral vasodilation and blood flow.

Photoacoustic and photothermal detection of circulating tumor cells, bacteria and nanoparticles in cerebrospinal fluid in vivo and ex vivo.

Circulating cells, bacteria, proteins, microparticles, and DNA in cerebrospinal fluid (CSF) are excellent biomarkers of many diseases, including cancer and infections. However, the sensitivity of existing methods is limited in their ability to detect rare CSF biomarkers at the treatable, early-stage of diseases. Here, we introduce novel CSF tests based on in vivo photoacoustic flow cytometry (PAFC) and ex vivo photothermal scanning cytometry. In the CSF of tumor-bearing mice, we molecularly detected in vivo circulating tumor cells (CTCs) before the development of breast cancer brain metastasis with 20-times higher sensitivity than with current assays. For the first time, we demonstrated assessing three pathways (i.e., blood, lymphatic, and CSF) of CTC dissemination, tracking nanoparticles in CSF in vivo and their imaging ex vivo. In label-free CSF samples, we counted leukocytes, erythrocytes, melanoma cells, and bacteria and imaged intracellular cytochromes, hemoglobin, melanin, and carotenoids, respectively. Taking into account the safety of PAFC, its translation for use in humans is expected to improve disease diagnosis beyond conventional detection limits.

Delivery of ion channel genes to treat cardiovascular diseases.

Modifying ion channel expression and function in the heart and vasculature are potentially useful, novel approaches to managing cardiac hypertrophy, atrial fibrillation and hypertension. Calcium channels play a pivotal role in the heart and vasculature in controlling muscle contraction as well as other aspects of calcium-dependent signaling. The present investigation reports development of mutated L-type calcium channel beta subunits that are delivered by an adenoviral vector to vascular smooth muscle tissue. Wild type subunits serve a chaperone function for the pore-forming alpha(1C) subunit of the calcium channel, localize to the cell membrane and enhance calcium current. Conversely, mutated subunits function as dominant negative, defective chaperone molecules that disrupt targeting to the cell membrane and decrease calcium current. The dominant negative genes can be delivered in vitro and ex vivo, and have the potential to decrease arterial tone and lower blood pressure in vivo.

Design of mutant beta2 subunits as decoy molecules to reduce the expression of functional Ca2+ channels in cardiac cells.

Calcium influx through long-lasting ("L-type") Ca(2+) channels (Ca(V)) drives excitation-contraction in the normal heart. Dysregulation of this process contributes to Ca(2+) overload, and interventions that reduce expression of the pore-forming alpha(1) subunit may alleviate cytosolic Ca(2+) excess. As a molecular approach to disrupt the assembly of Ca(V)1.2 (alpha(1C)) channels at the cell membrane, we targeted the Ca(2+) channel beta(2) subunit, an intracellular chaperone that interacts with alpha(1C) via its beta interaction domain (BID) to promote Ca(V)1.2 channel expression. Recombinant adenovirus expressing either the full beta(2) subunit (Full-beta(2)) or truncated beta(2) subunit constructs lacking either the C terminus, N terminus, or both (N-BID, C-BID, and BID, respectively) fused to green fluorescent protein were developed as potential decoys and overexpressed in HL-1 cells. Fluorescence microscopy revealed that the localization of Full-beta(2) at the surface membrane was associated with increased Ca(2+) current mainly attributed to Ca(V)1.2 channels. In contrast, truncated N-BID and C-BID constructs showed punctate intracellular expression, and BID showed a diffuse cytosolic distribution. Total expression of the alpha(1C) protein of Ca(V)1.2 channels was similar between groups, but HL-1 cells overexpressing C-BID and BID exhibited reduced Ca(2+) current. C-BID and BID also attenuated Ca(2+) current associated with another L-type Ca(2+) channel, Ca(V)1.3, but they did not reduce transient Ca(2+) currents attributed to Ca(V)3 channels. These results suggest that beta(2) subunit mutants lacking the N terminus may preferentially disrupt the proper localization of L-type Ca(2+) channels in the cell membrane. Cardiac-specific delivery of these decoy molecules in vivo may represent a gene-based treatment for pathologies involving Ca(2+) overload.