bluuebunny/crossref_metadata_2025 huggingface dataset:
Implication of the Histone Deacetylase 6 in pulmonary arterial hypertension
Authors: BOUCHERAT Olivier , CHABOT Sophie , PAULIN Roxanne , TRINH Isabelle , BOURGEOIS Alice , POTUS François , LAMPRON Marie‐Claude , LAMBERT Caroline , BREUILS‐BONNET Sandra , PARADIS Renée , NADEAU Valérie , GONCAROVA Elena , PROVENCHER Steeve , BONNET Sébastien
Published: November 2023

RATIONALE

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease of complex etiology. Despite environmental stressful conditions, pulmonary artery (PA) smooth muscle cells (PASMCs) and endothelial cells (PAECs) exhibit a pro‐proliferative and anti‐apoptotic phenotype. Histone deacetylase 6 (HDAC6) is a cytoplasmic histone deacetylase overexpressed in response to stress and implicated in the regulation of multiple pro‐survival mechanisms in cancer cells. Therefore, we hypothesized that HDAC6 expression is increased in PAH‐PASMCs and PAH‐PAECs allowing them to survive and proliferate, thus contributing to vascular remodeling in PAH.

OBJECTIVE

Using a multidisciplinary and translational approach we aimed to demonstrate that HDAC6 inhibition is a promising strategy to improve PAH.

METHODS AND RESULTS

HDAC6 is significantly up‐regulated (immunoblot) in lungs, distal PAs, and isolated PASMCs and PAECs from 10 PAH patients and 15 experimental PAH animal (5 Sugen/hypoxia rats; 5 monocrotaline rats and 5 chronic hypoxic mice) compared to controls. Molecular (siRNA) and pharmacological inhibition (Tubastatin and ACY‐775) of HDAC6 reduces dose‐dependently PAH‐PASMC/PAEC proliferation (Ki67 assay) and resistance to apoptosis (Annexin V assay) in vitro sparing control cells. Mechanistically, we demonstrated that HDAC6 deacetylates Ku70, blocking the translocation of Bax to mitochondria and preventing apoptosis. In vivo inhibition of HDAC6 (Tubastatin A 25mg/kg/day for 2 weeks) significantly improved (n=5 to 10 p<0.05) established PAH by decreasing mean PA pressure, total pulmonary resistance and increasing cardiac output in two experimental models (Sugen/hypoxia and monocrotaline). In addition, we showed that HDAC6 inhibitor can be safely given in combination with currently approved PAH therapies (macitentan and tadalafil). Finally, Hdac6 K.O mice have significantly lower right ventricle systolic pressure in response to 3 weeks of chronic‐hypoxia compared to wild‐type mice.

CONCLUSION

We showed for the first time that HDAC6 is implicated in PAH development and represents a new promising therapeutic target to improve PAH.

Support or Funding Information

None
Implication of the transcription Factor FOXM1 in pulmonary arterial hypertension
Authors: BOUCHERAT Olivier , TRINH Isabelle , LAMBERT Caroline , POTUS François , BREUILS‐BONNET Sandra , NADEAU Valérie , PAULIN Roxanne , PROVENCHER Steeve , BONNET Sébastien
Published: November 2023

RATIONALE

Pulmonary Arterial Hypertension (PAH) is a disease of the pulmonary vasculature, defined by an elevated pulmonary vascular resistance, leading to right heart failure and premature death. PAH is characterized by enhanced pulmonary artery smooth muscle (PASMC) proliferation and suppressed apoptosis within pulmonary artery (PA) wall. Targeting the cancer‐like of PASMCs represents an attractive therapeutic avenue to tackle PAH. Overexpression of the transcription factor FOXM1 has been shown to be a key driver of cancer progression through the stimulation of DNA repair, cell proliferation and cell survival.

OBJECTIVE

Using a multidisciplinary and translational approach we aimed to demonstrate that up‐regulation of FOXM1 in PAH‐PASMCs triggers proliferation and resistance to apoptosis.

METHODS AND RESULTS

FOXM1 is significantly increased (immunoblot and immunofluorescence) in lungs, distal PAs, and isolated PASMCs from PAH patients as well as in the monocrotaline (MCT)‐induced PAH model compared to controls. We demonstrated that, in response to growth factors like PDGF and IGF1, Akt is activated (increased P‐Akt), phosphorylates FOXO3a and inhibits it, allowing the up‐regulation of FOXM1. Moreover, we found that inhibition of FOXM1 in PAH‐PASMCs increases DNA damage (gH2AX) and reduces expression of NBS1 and PLK1 (immunoblot), two factors up‐regulated in PAH‐PASMCs (immunoblot and immunofluorescence) and involved in DNA repair and cell cycle progression. Consistently, pharmacological inhibition of FOXM1 using Thiostrepton dose‐dependently reduces PAH‐PASMC proliferation (Ki67 labeling) and resistance to apoptosis (Annexin V assay) in vitro. Inhibition of FOXM1 with Thiostrepton improves established PAH (right heart catheterization) in the MCT model.

CONCLUSION

We showed for the first time that FOXM1 is overexpressed in human PAH and implicated in the pro‐proliferative and anti‐apoptotic phenotype of PAH‐PASMCs.

Support or Funding Information

None
Reduced Membrane Cholesterol Following Chronic Hypoxia Limits Orai1 ‐Mediated Pulmonary Endothelial Ca2+ Entry
Authors: Zhang Bojun , Naik Jay S. , Jernigan Nikki L. , Walker Benjimen R. , Resta Thomas C.
Published: November 2023

Diminished Ca2+‐dependent production of endothelium‐derived vasodilators like nitric oxide is observed in chronic hypoxia (CH)‐induced pulmonary hypertension. Our previous studies show that endothelial store‐operated calcium entry (SOCE) as well as membrane cholesterol (Chol) are reduced in pulmonary arteries from rats exposed to CH (4 wk, 380 Torr) compared to their normoxic controls. Interestingly, Chol supplementation restores both membrane Chol and endothelial SOCE in cells from CH rats. Substitution of membrane Chol with epicholesterol (EpiChol, epimeric form of Chol) in freshly isolated pulmonary artery endothelial cell (PAEC) sheets mimics the effect of CH to blunt endothelial SOCE. These data suggest that reduced membrane Chol following CH limits the activity of ion channels required for endothelial SOCE. However, the ion channels involved in Chol‐sensitive SOCE have not been identified. We hypothesized that membrane Chol facilitates Orai1‐mediated endothelial Ca2+ entry, and that reduced Ca2+ entry following CH is due to loss of membrane Chol. To test this hypothesis, we pretreated PAEC sheets isolated from control and CH rats with Chol (2 mM) or EpiChol (2 mM), and administered the Orai1 inhibitor AnCoA4 (20 μM), or vehicle. Ca2+ influx in response store depletion with cyclopiazonic acid (CPA, 10 μM) was assessed by Mn2+‐quenching of fura‐2 fluorescence. Whereas Chol supplementation restored diminished SOCE in PAEC sheets from CH rats, both EpiChol and AnCoA4 significantly reduced endothelial SOCE in normoxic controls, but not in cells from CH rats. However, the Orai1 inhibitor had no further effect in cells pretreated with EpiChol. Additional experiments examined SOCE following Orai1 knockdown using siRNA in cultured rat pulmonary microvascular endothelial cells (PMVEC). We found that both EpiChol and Orai1 siRNA significantly inhibited SOCE compared to vehicle or non‐targeting siRNA, respectively. EpiChol treatment had no additional effect in cells following knockdown of Orai1. Furthermore, using a proximity ligation assay, we observed that EpiChol treatment significantly reduced the STIM1‐Orai1 interaction required for SOCE when compared to either vehicle or Chol treated groups. We conclude that membrane Chol is required for STIM1‐Orai1 interaction necessary to elicit endothelial SOCE. Furthermore, reduced Ca2+ entry after CH is due to loss of membrane Chol thereby limiting Orai1 activity.

Support or Funding Information

NIH R01 HL132883, NIH R01 HL111084, AHA 16GRNT27700010, AHA 15GRNT21080001,
Apoptosis in Pulmonary Hypertension is Regulated by the Sodium‐Hydrogen Exchanger
Authors: Huetsch John , Yun Xin , Suresh Karthik , Jiang Haiyang , Shimoda Larissa
Published: November 2023

Objective

Remodeling of the pulmonary vasculature, involving resistance to apoptosis by pulmonary arterial smooth muscle cells (PASMCs) and lung microvascular endothelial cells (LMVECs), is a key component of the pathology underlying pulmonary hypertension (PH). To better understand the mechanisms contributing to apoptotic resistance in PH, we studied endoplasmic reticulum (ER) stress signaling in PASMCs and LMVECs isolated from the SU5416‐hypoxia (SuHx) rat model of PH.

Methods

PH was induced in male Wistar rats via exposure to the vascular endothelial growth factor receptor inhibitor SU5416 plus 3 wk of 10% oxygen followed by 2 wk of normoxia (SuHx model), while control rats were exposed to vehicle and 5 wk of normoxia. PASMCs and LMVECs were isolated from SuHx and control rats and expanded in culture. Apoptosis was measured using Hoechst stain. Thioflavin fluorescence was quantified as a measure of ER stress. Protein levels of the ER stress‐induced transcription factor, CCAAT/enhancer‐binding protein homologous protein (CHOP), were measured via immunoblot. To measure activity of the sodium‐hydrogen exchanger (NHE), cells were incubated with pH‐sensitive fluorescent dye and the Na+‐dependent recovery of intracellular pH following acidification by ammonium pulse challenge was measured via fluorescence microscopy. Cell stress was induced with H2O2 (250 μM for PASMC; 100 μM for LMVEC). NHE was pharmacologically inhibited with 10 μM ethyl‐isopropyl amiloride.

Results

ER stress, which often induces apoptosis, was increased at baseline in PASMCs/LMVECs from SuHx rats. In control cells, H2O2 increased ER stress. Paradoxically, PASMCs and LMVECs from SuHx rats were protected from apoptosis both at baseline and following exposure to H2O2. The expression of CHOP, which is induced by ER stress and mediates apoptosis, was induced by H2O2 in control cells, but remained low in SuHx PASMCs/LMVECs. NHE activity was increased in PASMCs/LMVECs from SuHx rats, and NHE inhibition increased both basal and H2O2‐induced CHOP expression and apoptosis in SuHx PASMCs/LMVECs.

Conclusions

Decreased CHOP expression in the pulmonary vasculature during PH prevents ER stress‐induced apoptosis. Increasing CHOP expression via NHE‐inhibition restores stress‐induced apoptosis in PH vascular cells, suggesting a novel mechanism to control vascular remodeling in PH.

Support or Funding Information

NIH K08HL133475, F32HL124727, & R01HL073859
Studies on the Physical Properties of the Pseudomonas aeruginosa‐induced Endothelial Prion Amyloid Cytotoxin(s)
Authors: Morrow Kyle Adam , Stevens Trevor C , Agwaramgbo Ezinne , Langham Geri , Francis Michael , Balczon Ron , Stevens Troy
Published: November 2023

Rationale

Pseudomonas aeruginosa lung infection targets capillary endothelium and induces the production of prion amyloid cytotoxin(s), including a high molecular weight tau. These amyloid proteins are heat stable, RNase and DNase insensitive, and protease resistant. They are self‐replicating and transmissible among naïve cells. The physical nature of these amyloid cytotoxins remains incompletely resolved. Here, we examined the amyloid cytotoxin's angular velocity, and determined its degree of protease sensitivity and responsiveness to inactivation by post‐translational modification.

Methods

A P. aeruginosa strain, PA103, was used to infect pulmonary microvascular endothelial cells (PMVECs) for 4 hours, at which time cell supernatant was collected, centrifuged (4,500 × g for 10 minutes at room temperature), and filter sterilized (0.22 μm filter). Supernatant was divided into aliquots for centrifugation and sedimentation experiments, for protease treatment, and for post‐translational modification using diethylpyrocarbonate (DEPC). Centrifugation experiments were conducted at 150,000 × g (4°C) over a 24‐hour time course. Supernatant was exposed to proteinase K (100 and 500 μg/mL) digestion for 24 hours. DEPC (1, 10 and 20 mM) was incubated with supernatant for 30 minutes at room temperature for post‐translational modification of histidine residues. After these independent treatments, supernatant was added to PMVECs and gap formation and cytotoxicity (e.g. LDH release assay) determined. We used a custom plugin for ImageJ (NIH) software to automatically quantify intercellular gaps in images. The ratio of black to white pixels within each image was then measured to determine cell gap area.

Results

Supernatant samples from 0 (e.g. control), 1, 2, 4, 8, 16 and 24 hours of centrifugation were placed on PMVECs, and gap formation and cytotoxicity were measured. 0–4 hours centrifugation did not reduce cytotoxicity, whereas 8–24 hours centrifugation nearly eliminated cytotoxicity. Calculation of the angular velocity revealed the endothelial‐derived cytotoxin is small and/or less dense (1.14 × 1012) than the scrapie agent (3 × 1010), consistent with identification of a novel amyloid cytotoxin. The endothelial cytotoxin was extremely protease insensitive, as 24‐hour treatment with proteinase K was insufficient to eliminate its cytotoxicity. However, treatment with DEPC (10 mM), which post‐translationally modifies histidine side chains, eliminated cytotoxicity of the endothelial amyloid cytotoxin, similar to its effect on the scrapie agent.

Conclusion

P. aeruginosa infection of pulmonary endothelial cells causes production of a novel prion amyloid cytotoxin that is protease resistant and sensitive to post‐translational modification using DEPC.

Support or Funding Information

National Institutes of Health HL66299 and HL60024, American Heart Association 16POST27250139
Novel Role of Importins in Endothelial Permeability and Inflammation
Authors: Leonard Antony , Fazal Fabeha
Published: November 2023

Endothelial cell (EC) permeability and inflammation are central events in the pathogenesis of many inflammatory diseases including acute lung injury (ALI). Although importins (α and β) have been implicated in mediating the activation of NF‐kB, a master regulator of inflammation in epithelial and cancer cell, their role in regulating EC inflammation and barrier disruption remains unclear. In this study, we addressed the role of importins α4 and α3, adaptor molecules that interact with nuclear localization signal (NLS)‐containing proteins and importin β1, a carrier protein, which mediates the docking of the cargo/importin α complex to the nuclear pore to promote their translocation across the nuclear envelope, in the mechanism of EC permeability and inflammation. Exposure of EC to thrombin, a procoagulant and proinflammatory mediator, resulted in activation of the transcription factor NF‐κB and expression of its target genes VCAM‐1 and IL‐6. RNAi‐mediated knockdown of α4, α3 and β1 inhibited these responses. Further mechanistic analysis revealed that knockdown of α4, α3 or β1; each inhibited thrombin‐induced DNA binding activity of RelA/p65 as well as its phosphorylation at Ser536, a critical event that controls transcriptional capacity of RelA/p65 bound to DNA. Intriguingly, knockdown of α3, α4 or β1, each protected against thrombin‐induced EC barrier disruption. Together these studies identify a novel role of α3, α4 and β1 in EC inflammation and permeability associated with intravascular coagulation.

Support or Funding Information

NIH (HL096907 and HL130870)
From Field to Lab: Asking Questions about Hypoxia Regulation of Vascular Tone in Cell Culture Systems
Authors: Hindle Allyson , Allen Kaitlin , Batten Annabelle , Wareham Lauren , Buys Emmanuel , Zapol Warren
Published: November 2023

Seals are physiologically specialized to exploit prey resources below the ocean surface through extended breath‐hold diving. This dive response includes remarkable cardiovascular control, managed regional blood flow based on metabolic need, and an ability to sustain periods without oxygen. To identify the molecular mechanisms that underlie the seal's natural protection of critical tissues by region‐specific control of perfusion, we investigated the nitric oxide (NO) – cyclic GMP system in the vasculature of Weddell seals. In terrestrial mammals, this pathway responds to tissue hypoxia to trigger local vasodilation. Underwater, any tissue may experience hypoxia, yet perfusion remains under tight regional control. We assayed cGMP production in response to stimulation by NO donors in vitro. In intact arteries collected from animals at necropsy, preliminary results indicate that NO‐cGMP signaling may be impaired in Weddell seals compared to terrestrial mammals, however, the system remains functional in all vascular beds examined. To complement this in vitro assessment of gene expression and enzyme activities in key pathway enzymes, we established cultured endothelial cells from placental arteries in the field. After return of cryo‐preserved primary cells to the US, they were tested for expression of cell type‐specific enzymes, then experimentally perturbed to either stimulate/repress NO‐cGMP signaling or to provide hypoxia exposure. These cell resources provide an opportunity to address mechanistic questions about free‐living animals in the lab, and their phenotype can be validated through comparisons with intact tissue samples. Because diving ability defines and limits the role of marine mammals in their ecosystem, we can apply a mechanistic understanding of the dive response to understand the ability of these species to respond to natural and anthropogenic disturbance. In future, these insights also have tremendous potential for human medicine, pointing to novel therapies for cardiovascular trauma (stroke, heart attack) and diseases associated with tissue hypoxia (pneumonia, sepsis, cancer).

Support or Funding Information

Funding by NSF Division of Polar Programs
HIF‐1 plays a key signaling role in physiological responses to acute hypoxia in the African naked mole rat, Heterocephalus glaber
Authors: Pamenter Matthew E
Published: November 2023

Naked mole rats are among the most hypoxia‐tolerant mammals identified. This tolerance is partially achieved by their ability to coordinately reduce metabolic and ventilatory rates by > 85% in acute hypoxia. Our understanding of the physiological mechanisms that enable their hypoxia‐tolerance is improving; however, the cellular mechanisms that mediate these responses remain poorly understood. Hypoxia‐inducible factor‐1α (HIF‐1α) is a transcription factor that is central to hypoxic signaling at the cellular level. In most mammals, HIF‐1α is degraded in normoxia but is stabilized when oxygen levels are reduced. Stabilization of HIF‐1α leads to upregulation of a multitude of genes that promote survival in hypoxia. A recent analysis of the naked mole rat genome revealed several mutations to HIF‐1α. These mutations are predicted to prevent HIF‐1α degradation during normoxia, which would chronically upregulate HIF‐1α‐mediated signaling pathways. We hypothesized that endogenous activation of HIF‐1α‐mediated signaling contributes to the remarkable hypoxia‐tolerance of naked mole rats by modulating their metabolic rate. To test our hypothesis, we used pharmacological tools to manipulate HIF‐1α expression in naked mole rats and mice and measured metabolic and ventilatory responses to an acute hypoxic challenge (7% O2 for 1hr) in awake and unrestrained subjects using plethysmography and respirometry. Pharmacological manipulation of HIF‐1α expression was confirmed by measuring changes in the expression of several downstream gene targets of HIF‐1α. In naked mole rats, blocking HIF‐1α had only minor effects on ventilation but drastically blunted the hypoxic metabolic response. As a result, the naked mole rat air convection requirement (ACR) increased 2‐fold in normoxia and 3‐fold in hypoxia, indicating that naked mole rats were hyperventilating and thus less tolerant of hypoxic stress. Conversely, activating HIF‐1α in mice reduced ventilation and metabolic rate in both normoxia and hypoxia and also reduced the hypoxic increase in the murine ACR, indicating that HIF‐1α‐agonist‐treated mice were more tolerant of hypoxic stress. We conclude that endogenous upregulation of HIF‐1α signaling contributes to the hypoxia‐tolerance of naked mole rats, and we demonstrate that this mechanism can be translated to hypoxia‐intolerant mice to induce a hypoxia‐tolerant phenotype.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada, Canada Research Chairs Program, Parker B Francis Foundation
Developmental differences in cardiac transcriptome suggest gene candidates underlying differences in anoxia tolerance in the Western Painted Turtle
Authors: Fanter Cornelia E , Warren Daniel E
Published: November 2023

Adult Western painted turtles, Chrysemys picta bellii, are the most anoxia‐tolerant tetrapod known and is capable of surviving over 170 days of anoxia at 3°C. Hatchling turtles, however can survive only 41 days at 4°C. To investigate the transcriptomic basis for this difference, we compared the anoxia‐induced transcriptional responses in the ventricle of adult and hatchling painted turtles exposed to four different conditions: 20 °C acclimation, five weeks at 3 °C, 20 days of anoxia at 3 °C, and re‐exposure to oxygen at 3 °C. At each time point, adult and hatchling turtles were sampled and RNA extractions were performed on the frozen, homogenized ventricular tissue. Illumina HiSeq 4000 was used to sequence mRNA, and transcript expression levels were compared between treatments to determine the changes attributed to temperature, anoxia exposure and development.

Support or Funding Information

This work was supported by a CAREER award from the National Science Foundation.
Mechanisms of increased lifespan in hypoxia
Authors: Greenlee Kendra J , Bowsher Julia H , Heidinger Britt , Rinehart Joseph P , Yocum George D
Published: November 2023

Genetic variation accounts for a relatively small amount of the variation in lifespan (generally 15–30%), while environmental stressors are very strong predictors (Finch and Kirkwood 2000). Hypoxia is an environmental stress that increases longevity in some contexts, but the mechanisms remain poorly understood. Our long‐term goal is to understand the mechanisms by which hypoxia extends lifespan. In the bee Megachile rotundata, lifespan doubles upon rearing in hypoxia (Abdelrahman et al. 2014). We hypothesize that that hypoxia increases longevity by limiting telomere loss from reactive oxygen species (ROS). In this study, prepupal bees were maintained in 10, 21 or 24% oxygen for 9 months. Each month, two subsets of bees were removed. One group was immediately frozen for processing. A second subset of bees was reared to adulthood and then frozen for processing. Similar to our previous findings, all bees had extended lifespans compared to bees in the wild, but bees exposed to hypoxia had high survival after 9 months in 10% oxygen. At nine months, bees reared in 24% oxygen had 14% survival, while those in normoxia had 41% survival. Because they survived better than bees in normoxia or hyperoxia, we predicted that bees in hypoxic conditions would have reduced telomere loss rates, lower levels of ROS, and/or higher levels of telomerase activity than bees exposed to normoxia. We measured total antioxidant activity and found no differences among treatments from either prepupal or adult bees, indicating that oxidative stress is not regulated at the level of antioxidants. Because antioxidants are invariant, we predict that increased oxidative damage to proteins will be observed under normoxic and hyperoxic conditions. We expect bees reared in hypoxia to have longer telomeres than bees reared in normoxia or hyperoxia. In addition, we expect bees reared in hypoxia to have lower levels of oxidative damage than bees reared in normoxia or hyperoxia.

Support or Funding Information

NIH COBRE to NDSU, USDA‐ARS
Prenatal Thyroxine Treatment promotes Anxyolisis in Swiss Mice Offspring
Authors: Conceição Rodrigo Rodrigues , Laureano‐Melo Roberto , Souza Janaina Sena , Albuquerque Josie Marcelle Marcelle Lira , Giannocco Gisele , Olivares Emerson Lopes , da Silva Cortes Wellington
Published: November 2023

The proper functioning of the maternal thyroid plays a crucial role in fetal development. Thus, the aim of our study was to verify how maternal hyperthyroidism is able to change behavioral parameters in mice's offspring during adulthood. For this purpose, two groups (n=6 each, and ~35g) of pregnant Swiss mice were randomly divided into two groups: control and thyroxin‐treated (T4) group. The control was treated with 0.9% saline, while the treated group received T4 (200μg/kg, s.c.) during all pregnancy period. By completing 70 days of life, the puppies were underwent to a battery of tests in the following sequence: open field, dark‐light box, elevated plus maze, marble burying, rotarod and tail suspension test. At the end of the experiment, hippocampus was dissected for RNA analysis. Statistical analysis was performed by t‐Student and the means were considered significantly different when p<0.05. According to our results, it was shown that T4 group had an increase of 52.42 % in total distance traveled (16.31± 1.04 m vs. 10.7 ± 0.68 m, p < 0.001) in open field test. It was also shown difference of 13.62% in light time side (126.8 ± 3.10 sec. vs. 111.6 ± 3.59 sec., p = 0.004) in light‐dark box paradigm. In elevated plus maze test, we observed a higher time (42.09 ± 4.29 sec. vs. 24.64 ± 6.00 sec., p = 0.02) and percentage of entries (27.88 ± 3.33 vs. 15.56 ± 2.47, p = 0.008) in the opened arms. In hippocampus, T4 offspring had higher expression of TPH2 (4.26 ± 0.71 vs. 1.33 ± 0.49, p = 0.01), SERT (3.92 ± 0.78 vs. 0.56 ± 0.18, p = 0.005), 5HT1a (1.80 ± 0.18 vs. 1.10 ± 0.19, p = 0.03) and GAD 67 (2.62 ± 0.40 vs. 1.18 ± 0.35, p = 0.02). These findings indicate that gestational hyperthyroidism alters hippocampus serotonergic and gabaergic systems, reducing anxiety‐like behavior in adults.

Support or Funding Information

FAPERJ & CNPq
From vision to action: the planning and execution of eye movements
Authors: Smith Matthew A , Khanna Sanjeev B , Snyder Adam C
Published: November 2023

As we navigate the visual world we move our eyes about in a ballistic fashion roughly three times every second. This process, known as a saccade, brings the high‐resolution portion of our retina, the fovea, onto an object in the visual world. Circuits involved in the planning and execution of eye movements have been well studied across the brain, especially the brain stem circuits that lead to contraction and relaxation of the extraocular muscles. Relatively less is known about the transition from visual inputs to eye movements, a process that involves the cerebral cortex. In particular, a portion of prefrontal cortex known as the frontal eye fields (FEF) have been implicated in the cortical control of eye movements. This brain region contains visual neurons that respond to a flash of light and motor neurons that respond just prior to an eye movement. This is an ideal locus for studying the visuomotor transformation, because both types of neurons exist in close proximity and because one of the outputs of FEF is a set of descending projections that lead to the movement itself. We hypothesized that populations of visual and motor neurons in FEF were functionally organized in a manner that efficiently transforms visual information into a motor command. We studied this by performing extracellular recordings of populations of FEF neurons using a linear electrode array in alert rhesus macaque monkeys performing a conventional memory guided saccade task in which they made an eye movement to the location where a visual stimulus had previously appeared. This task permits evaluation of the visual response, the motor response, and the delay period in between when the eye movement plan must be generated from the sensory stimulus. We focused on the correlated variability among neurons, because it is a key determinant of how well their activity could be decoded by downstream neurons that drive the eye movement itself. When we analyzed the connections between visual and motor neurons, we found a distinct pattern of results. The overall level of correlated variability between these groups was lower than visual‐visual or motor‐motor pairs, and it changed dramatically depending on the direction of the planned eye movement. A decoding analysis revealed that these changes with eye movement direction were organized in a fashion that would be advantageous to downstream neurons in executing the eye movement. These findings suggest that visual and motor populations of neurons in FEF play a unique role in transforming visual information to motor output.

Support or Funding Information

NIH Grants EY022928 and EY008098
Persistent pain intensifies the recall of already consolidated fear memories
Authors: Dimitrov Eugene
Published: November 2023

The deleterious impact of chronic pain on learning, memory and mood is well documented. Long‐lasting pain leads to anxiety, depression and negatively impairs learning and formation of new memories. We hypothesized that chronic pain may affect even memories that were established before the onset of pain. Mice underwent a classic auditory fear conditioning that included 30 seconds 75 dB tone as a conditioned stimulus (CS) that was paired with 2 seconds 1 mA electric foot shock as unconditioned stimulus (US). Sciatic nerve constriction by inserting a small plastic tube around the left sciatic nerve was used to induce neuropathic pain 48 hours after the fear conditioning. This nerve constriction, also called “cuffing”, reliably produced mechanical allodynia that lasted for the duration of the experiments. Fear recall was measured by exposing the mice to the CS and recording their activity for five minutes. The time mice were completely immobile (freezing time) was expressed as a percent. Two sessions of fear recall were conducted, one 14 days and second 28 days after the initiation of neuropathic pain. Mice with neuropathic pain showed an increased percent of time freezing to CS (sham group with 22.7% ± 1.4 versus an average of 38.2% ± 4.3 freezing time for the pain group, t36 =3.9, P<0.001). The cuffed mice once again spent more time freezing when retested 28 days after the beginning of pain (the average freezing time was 22.9% ± 2.9 for the shams versus 49.5% ± 4.9 of the cuffed group, t36= 4.8, P< 0.001). In the next experiment the sciatic nerve constriction was applied two weeks after fear conditioning. These mice showed increased fear response to the CS 14 days after the onset of pain or 28 days after the fear conditioning took place (the freezing time of the sham group was 44.6% ± 6.5 versus the cuffed group with 69.5% ± 5.4 freezing time, t18 = 2.9, P<0.001). When tested for anxiety and depression, the experimental groups did not display any increased anxiety‐like or depression‐like behavior in comparison to the control groups. However, the Sholl analysis of the pyramidal neurons in basolateral amygdala of mice with neuropathic pain showed increased dendritic length (the average dendritic length was 701.9 μm ± 40.1 in sham group versus 955.7 μm ± 46.2 in cuffed group, df25=2.7, P<0.02).

In conclusion, our studies provide evidence for impact of persistent pain on already consolidated fear memories. Very likely the underlying mechanism for this phenomenon is increased dendritic plasticity of the pyramidal neurons brought by overall decreased inhibitory tone in basolateral amygdala during persistent pain. It is possible that the increased fear recall to already consolidated fear memory is a harbinger for a later development of anxiety and depression symptoms that are typical for chronic pain.

Support or Funding Information

Grant # 5R01MH105528‐04
The Nuclear Receptor REV‐ERBα Alters Mood and Neurotransmission in the Prefrontal Cortex
Authors: Otsuka Tsuyoshi , Kohsaka Akira , Le Hue Thi , Nakao Tomomi , Thein Zaw Lin , Maeda Masanobu
Published: November 2023

A defect in behavioral rhythms is a key feature of mood disorders such as depression, seasonal affective disorder, and bipolar disorder. Recent studies have proposed REV‐ERBα as a key circadian nuclear receptor that links behavioral rhythms to mood regulation. However, how REV‐ERBα regulates mood in the brain remains to be elucidated. Here we show that REV‐ERBα is abundantly expressed in neuronal cells and that the prefrontal cortex expresses Rev‐erbα at the highest level among mood‐controlling areas of the brain. To further explore the role of REV‐ERBα in the regulation of mood, we analyzed transcriptome in the prefrontal cortex of Rev‐erbα deficient mice. Importantly, we found marked alteration in the expression levels of a cluster of genes known to regulate mood such as Fabp7, Bdnf, and Nr4a1 in the prefrontal cortex of Rev‐erbα deficient animals. We also found that deficiency in the Rev‐erbα gene altered expression levels of genes associated with neuronal activity and plasticity in the prefrontal cortex. More importantly, a quantitative UPLC assay using tissues of the prefrontal cortex without the Rev‐erbα gene detected changes in the levels of key neurotransmitters which can directly affect mood regulation. Taken together, our results suggest that the circadian nuclear receptor REV‐ERBα plays an important role in the regulation of neurotransmission in the prefrontal cortex that coordinates mood and behaviors.

Support or Funding Information

This work was supported by JSPS KAKENHI Grant Number 16K19781
Heat Exposure Improves Cognitive Performance in Persons with Higher Levels of Spinal Cord Injury
Authors: Handrakis John P , Tittley Tishina D. , Tascione Oriana F. , Bart Jessica , Barton Christian , Bang Charlene , Spungen Ann M. , Bauman William A.
Published: November 2023

Our previous work demonstrated that core body temperature (Tcore) is typically lower in persons with tetraplegia and that limited exposure to cool temperature results in a decline in Tcore with an associated decline in cognitive performance, while a similar cool exposure in able‐bodied (AB) individuals did not affect Tcore or cognitive performance. Because of the prior observed effect of cool exposure in those with tetraplegia, the effect of heat exposure on Tcore and cognitive performance was determined in persons with higher spinal cord lesions (above T4).

Fifteen persons with spinal cord injury (SCI) (C5‐T3, AIS A‐B) and 15 age and body mass index‐matched AB controls were acclimated to 27°C at baseline (BL) before being exposed to 35°C for up to 120 minutes (Heat Challenge). Tcore (rectal temperature) was continuously measured while cognitive performance was assessed at BL and at the end of Heat Challenge using Stroop Color and Word and Wechsler Adult Intelligence Scale‐Fourth Edition (WAIS‐IV) Digit Span tests. Persons with SCI were divided into two groups based on the magnitude of change in Tcore to Heat Challenge, High Responder (HR: > 0.5°C) and Low Responder (LR: <0.5°C).

At BL, average Tcore was 36.7±0.43°C in HR (9 participants with C5‐T1), 37.2±0.33°C in LR (6 participants with C5‐T3), and 37.3±0.36°C in AB controls; Tcore was different between HR and AB groups (p<0.05). After Heat Challenge, Tcore was not significantly different among the three groups with Tcore increasing 0.75±0.32°C in HR, 0.27±0.16°C in LR, and −0.05±0.25°C in AB groups; the changes in Tcore after challenge were significant only in the HR and LR groups. On measures of cognitive performance, HR group had a significantly greater percent change compared to AB group in Stroop Word (19.8±13.5% vs. 5.2±13.9%; p=0.02, respectively) and WAIS‐IV Sequence (24.1±17.2% vs. −2.2±19.5%; p=0.003, respectively); no differences in cognitive performance were found between the LR and AB groups.

Those persons with higher levels of SCI who had a greater rise in Tcore after a thermal challenge and a lower Tcore at BL also demonstrated improved cognitive performance. Thus, the HR group displayed a greater impairment in thermoregulation with associated increased vulnerability to exposure to extremes in ambient temperature. Because subnormal Tcore has been demonstrated to impair cognitive performance, efforts to maintain euthermia in vulnerable persons with higher levels of SCI should be considered with the use of practical and effective strategies to accomplish this objective.

Support or Funding Information

VA Rehabilitation Research and Development Service, Small Projects in Rehabilitation Research (SPiRE): Federal Award Identification Number: I21RX001734
A Triple Combination Approach Involving Nerve Transplantation, Glial Scar Digestion and Passive Exercise Promotes Cardiovascular Recovery after Spinal Cord Injury
Authors: Sachdeva Rahul , Gopaul Rayshad , Jia Mengyao , Monga Aaron , Ramer Matt , Krassioukov Andrei V.
Published: November 2023

Introduction

Cardiovascular (CV) dysfunction is the leading cause of morbidity and mortality after spinal cord injury (SCI). SCI causes CV dysfunction primarily by (a) the direct loss of critical medullary control of sympathetic pre‐ganglionic neurons and, (b) the aberrant sprouting of nociceptive afferent fibers within the spinal cord. To promote significant CV recovery after SCI, we developed a triple combination approach to simultaneously promote axonal regeneration to regain the supraspinal vasomotor control as well as to reduce aberrant sprouting of nociceptive afferent fibers in an experimental model of SCI.

Methods

Adult male Wistar rats (n=40) received a complete spinal transection (Tx) at 3rd thoracic segment. The treatment group received peripheral nerve grafts (PNGs) spanning the lesion, intrathecal chondroitinase enzyme (ChABC) and cycling exercise beginning on day 5 (1 hr/day, 5 days/week). Appropriate control groups received Tx only or Tx and a single intervention. At 8 weeks post injury, CV function [i.e. resting blood pressure (BP), heart rate and pressor response to colorectal distension (CRD)] was assessed using radiotelemetry. Functional regeneration across PNGs was determined using stimulus driven units below the lesion upon electrical stimulation at cervical level. Neural tract tracing and immunohistochemistry was performed to quantify the number of regenerating neurons as well as the sprouting of nociceptive (calcitonin gene related peptide positive) afferent fibers.

Results

The preliminary findings (n= 5/group) show that the combination approach (Tx+PNG+ChABC+Ex) leads to significant CV recovery compared to the untreated group (Tx alone). Specifically, in response to CRD, the treatment group shows 53.8% reduction in systolic BP elevation (77.8 vs. 37.7 mmHg, p<0.0001, Fig. A), 42.2% reduction in diastolic BP elevation (42.5 vs. 24.5 mmHg, p<0.001, Fig. B), 57.6% reduction in mean arterial pressure elevation (54.2 vs. 22.9 mmHg, p<0.001, Fig. C) as well as abolishment of CRD‐induced bradycardia (−80.2 vs. +14.6 bpm, p=0.056, Fig. D). Synaptically driven activity of spinal cord neurons at T4 level upon electrical stimulation above the injury confirmed the functional re‐connection of regenerating axons. Histological studies are underway to further investigate the regeneration/plasticity related mechanisms underlying recovery.

Conclusion

Combined neuro‐regenerative and rehabilitative approach leads to significant recovery of CV function after SCI, which is likely mediated by vasomotor regeneration and afferent plasticity manipulation.

Support or Funding Information

Present study was supported by the Rick Hansen Foundation start up funding and the Heart and Stroke Foundation of Canada (AVK).
Dapagliflozin, a Selective SGLT2 Inhibitor, Attenuated Cardiac Fibrosis by Regulating the Macrophage Polarization via STAT3 Signaling in Infarcted Rat Hearts
Authors: Lee Tsung‐Ming , Chang Tsang‐Wei , Yang Chen‐Chia
Published: November 2023

During myocardial infarction, delayed M1 toward M2 macrophage phenotype transition is considered one of the major factors for adverse ventricular remodeling. We investigated whether dapagliflozin, a sodium‐glucose cotransporter 2 (SGLT2) inhibitor, attenuates cardiac fibrosis via regulating macrophage phenotype by a reactive oxygen and nitrogen species (RONS)/STAT3‐dependent pathway in postinfarcted rats. Normoglycemic male Wistar rats were subjected to coronary ligation and then randomized to either saline, dapagliflozin (a specific SGLT2 inhibitor), phlorizin (a nonspecific SGLT1/2 inhibitor), dapagliflozin + S3I‐201 (a STAT3 inhibitor), or phlorizin + S3I‐201 for 4 weeks. There were similar infarct sizes among the infarcted groups at the acute and chronic stages of infarction. At day 3 after infarction, post‐infarction was associated with increased RONS levels, which can be inhibited by administering either dapagliflozin or phlorizin. SGLT2 inhibitors significantly increased STAT3 activity, STAT3 nuclear translocation, myocardial IL‐10 levels and the percentage of M2 macrophage infiltration. At day 28 after infarction, SGLT2 inhibitors were associated with attenuated myofibroblast infiltration and cardiac fibrosis. Compared with phlorizin, the extent of dapagliflozin towards M2 polarization was significantly higher. The effects of SGLT2 inhibitors on M2 polarization were nullified by adding S3I‐201. Furthermore, the effects of dapagliflozin on STAT3 activity and myocardial IL‐10 levels can be reversed by 3‐morpholinosydnonimine, a peroxynitrite generator. These observations provide a novel mechanism of SGLT2 inhibitors‐mediated M2 polarization through a RONS‐dependent STAT3‐mediated pathway and selective SGLT2 inhibitors are more effective in attenuating cardiac fibrosis during postinfarction remodeling.

Support or Funding Information

nil
Inhibition of Cyclophilin D During Acute and Chronic Myocardial Infarction in Rats: The Effect of Reperfusion
Authors: Parodi‐Rullán Rebecca Maria , Soto‐Prado Jadira , Vega‐Lugo Jesús , Chapa‐Dubocq Xavier , Javadov Sabzali
Published: November 2023

Background

Mitochondrial permeability transition pore (mPTP) opening plays a critical role in pathogenesis of myocardial infarction (MI) and cardiac ischemia‐reperfusion (IR). Cyclophilin D (CyP‐D) is the only protein which has been broadly accepted as a major regulator of the mPTP complex. Therefore, inhibition of CyP‐D is mostly used as a target for inhibition of mPTP opening and thus, prevention of mitochondria‐mediated cell death induced by MI and IR injury. In addition to mPTP regulation, CyP‐D also plays an important physiological role and controls cell metabolism through regulation of glucose uptake, fatty acid oxidation, and mitochondrial bioenergetics. Growing data on the physiological role of CyP‐D and mPTP opening as well as failure of recent CIRCUS (Cyclosporine and Prognosis in Acute Myocardial Infarction Patients) clinical trials when cyclosporin A (CsA) failed to protect the heart in STEM patients, indicates the necessity for further studies elucidating the role of CyP‐D inhibition to exert cardioprotection against IR injury. Here, we hypothesized that inhibition of CyP‐D may have different effects in non‐reperfused MI and reperfused MI (IR).

Methods

MI was induced in adult female rats (200–250g) by ligation coronary artery (CAL) for 30 min followed by 2‐ and 28‐day post‐surgery with or without reperfusion. The animals were studied in the following groups: i) Sham (S, no MI, n=6 and n=11 for 2 and 28 days, respectively); ii) SS (Sham+SfA, n=5 and n=11 for 2 and 28 days, respectively); iii) MI (no reperfusion, n=4 and n=5 for 2 and 28 days, respectively); iv) MI+SfA (n=4 and n=6 for 2 and 28 days, respectively); v) MI+R (MI+reperfusion, n=4 and n=5 for 2 and 28 days, respectively); and vi) MI+RS (MI+reperfusion+SfA, n=6 and n=7 for 2 and 28 days, respectively). SfA (5mg/kg, IV) was administered at 25 min after a start of sham surgery or MI. Cardiac function was analyzed at 2 and 28 days post‐surgery by echocardiography.

Results

SfA significantly improved cardiac function (ejection fraction, EF%) (33±5 vs. 53±4, P<0.01) after 2 days, but it had no beneficial effects at 28 days (38±3 vs. 42±4). Likewise, SfA administration to permanent occluded hearts has no effect on EF% at 2 (44±9 vs. 35±9) and 28 (37±3 vs. 44±5) days. SfA administration to control animals reduces cardiac EF% at 2 (69±3 vs. 52±3, P<0.001) and 28 days (66±2 vs. 55±3, P<0.01).

Conclusion

Our data indicates that inhibition of CyP‐D by SfA has cardioprotective effects on MI at early reperfusion and no effect on MI without short‐ or long reperfusion.

Support or Funding Information

Supported by NHLBI NIH (SC1HL118669) and NIH MBRS‐RISE Program (R25‐GM061838).
Reduced SIRT3 contributes to large elastic artery stiffness with aging
Authors: Brodjeski Alexander Lee , Schaeffer Gilbert V , Ear Po Hien , Brenner Charles , Sindler Amy L
Published: November 2023

Age‐related increases in arterial stiffness are mediated in part by mitochondrial dysfunction. Sirtuin 3 (SIRT3) is a mitochondrial NAD+‐dependent deacetylase that regulates mitochondrial function. SIRT3 deficiency contributes to physiological dysfunction in a variety of pathological conditions. Here, we tested the hypothesis that age‐associated arterial stiffness, assessed by aortic pulse wave velocity (aPWV), would be accompanied with decreased SIRT3 expression and activity. Old (~26 mo, n = 9) C57BL/6N male mice had higher aPWV vs. young (6 mo, n = 10) (448 ±14 vs 382 ± 13, p < 0.005), which was associated with reduced arterial and renal SIRT3 protein expression (0.17 ± 0.04 n=5 vs 0.57 ± 0.11 n=7, p<0.05; 0.70 ± 0.09 n=6 vs 0.96 ± 0.08 n=6, p =0.05, respectively). Aortic SIRT3 protein expression was negatively correlated aPWV (R=−0.7798, p<0.005). Furthermore, SIRT3 KO male mice demonstrated higher aPWV compared to age‐matched control mice (480±20.77 n=6 vs. 391.3±12.3 n=7, p<0.005). Downstream markers of SIRT3 regulatory activity were examined. Taken together, these data suggest that the age‐associated decline in SIRT3 contributes to increased arterial stiffness and may be a potential target for improving vascular function.

Support or Funding Information

Supported by NIH K01AG047626 and University of Iowa Department of Health and Human Physiology.