bluuebunny/crossref_metadata_2025 huggingface dataset:
Cardiac structure and function in mice with a cardiomyocyte‐specific knockout of Na+/K+‐ATPase α1‐isoform
Authors: Marck Pauline V , Wang Xiaoliang , Kutz Laura , Cui Xiaoyu , Blanco Gustavo , Cai Liquan , Xie Zijian , Pierre Sandrine V
Published: November 2023

Owing mostly to the clear impact of the Na/K‐ATPase specific ligand ouabain and other cardiac glycosides (CG) on cardiac contractility and myocyte size, the importance of both Na/K‐ATPase ion‐pumping and signaling functions in the regulation of cardiac structure and performance have been recognized for decades. In the mouse heart, about 80% of Na/K‐ATPase activity is carried on by α1‐containing enzyme complexes relatively insensitive to ouabain, and the remainder is supported by ouabain‐sensitive α2‐containing enzymes. Evidence obtained in mammalian cell models expressing either one of these isoforms suggest that they serve highly specific cellular functions, but it has been technically challenging to investigate them in a cardiac myocyte and assess the impact on cardiac structure and function. We generated a mouse model with a cardiomyocyte‐selective Na/K‐ATPase α1‐isoform knockout using α‐myosin heavy chain (Myh6) cre/loxP recombination. Animals with a cardiac α1−/− genotype (cardiac α1−/−) were born with the expected Mendelian frequency and survived to adulthood. Western blot analysis confirmed the absence of detectable α1 protein with an apparent compensatory 2.6 fold upregulation of α2 expression without altering Na/K‐ATPase β1 isoform expression in cardiac α1−/− hearts. No major changes in heart weight/body weight or heart weight/tibia length ratios were noted in a cohort of males at 4 month of age. Transthoracic echocardiography studies in isoflurane‐anesthetized animals did not reveal a substantial adverse impact on cardiac function in this cohort, and a mild but significant increased stroke volume was noted in cardiac α1−/− mice (+27%, 23 μl± 1 μl vs 29 μl± 1 μl, P<0.01, n=5–6). These results suggest that the cardiac α1−/− is viable and shall provide a useful model in which to study the role of Na/K‐ATPase α‐isoforms and the impact of cardiac Na/K‐ATPase signaling in health and diseases.

Support or Funding Information

Marshall Institute for Interdisciplinary Research and NIH Grant HL‐109015
Na/K‐ATPase α1 Isoform as a Critical Signal Integrator in Embryonic Development
Authors: Wang Xiaoliang , Zhang Jue , Cui Xiaoyu , Wang Jiayan , Cai Liquan , Pierre Sandrine V , Xie Zijian
Published: November 2023

We have shown that Na/K‐ATPase interacts with caveolin‐1 through a putative caveolin‐binding motif, and regulates its trafficking in cell culture. The aim of this study is to review the importance of this putative interaction in animal physiology. A mCBM (F97A and F100A) mutant mouse was generated using a knock‐in strategy. Homozygosity of the mCBM Na/K‐ATPase animal results in embryonic lethality around mid‐gestation. Heterozygous animals were born with the expected Mendelian frequency and survived to adulthood. Further analysis of the embryonic development reveals the following defects. The first phenotype of homozygous embryos with obviously defect was noted at E7.0 day. At E9.0 day, homozygous embryos exhibited severe growth retardation with defective brain and somite development. By E12.5 day, the homozygous embryos were no longer detectable. On the other hand, heterozygous embryos seems to be normal through the development. However, they are significantly smaller in size at E9.0 day. Mechanically, RNAseq shows normal expression of different isoforms of Na/K‐ATPase and caveolin‐1. However, it reveals defects in Wnting signaling pathway, which results in severe down‐regulation of genes important for neurogenesis and muscle genesis. Those defects were further confirmed by qPCR analysis of mRNA from wild type and homozygous embryos. Taken together, our new findings suggest that α1 Na/K‐ATPase functions as a critical signal integrator in animal embryonic development, and that the putative caveolin‐binding motif is required for this non‐pumping function of Na/K‐ATPase.

Support or Funding Information

Marshall Institute for Interdisciplinary Research and NIH Grant HL‐109015
Uptake and Protein Interaction of APOL1 with HSP60 in the Proximal Tubule
Authors: Blessing Natalya A , Konieczkowski Martha , Madhavan Sethu , O'Toole John , Sedor John , Bruggeman Leslie
Published: November 2023

Coding variants of APOL1, common in African Americans, have been linked to the elevated risks of chronic kidney disease (CKD) associated with African ancestry. While all variants of apolipoprotein 1 (APOL1) circulate in the serum bound to HDLs and confer protection against Trypanosoma brucei brucei, APOL1 G1 and G2 variants offer additional protection against T.b. rhodesiense. T.b. rhodesiense produces serum‐resistance associated (SRA) protein, which binds to the C‐terminal region of APOL1 G0 and blocks trypanolysis. APOL1 G1 and G2 evade the trypanolysis block through a reduced (G1) or nonexistent (G2) interaction with SRA. Preliminary studies have demonstrated that both APOL1 protein and mRNA are present in glomerular podocytes; however, mRNA in situ hybridization failed to detect APOL1 transcripts in the proximal tubule (PT), despite APOL1 protein being most abundant in the mitochondria of PT cells. APOL1 function in the context of kidney disease and the route of apparent uptake into the PT remains unknown. To investigate the renal function of APOL1, we hypothesized that APOL1 variant‐dependent interaction with SRA implies a variant‐dependent interaction between APOL1 and an SRA‐like binding partner in humans that contributes to APOL1‐associated CKD. To identify human APOL1‐interacting proteins, a yeast‐two‐hybrid screen of human cDNA library using the SRA‐interacting domain of APOL1 as bait detected heat shock protein 60 (HSP60), a stress response chaperone for mitochondrial proteins, as a binding partner for APOL1. Confirmatory yeast two hybrid and immunoprecipitation experiments observed differential binding of APOL1‐G0 compared to the CKD variants, G1 and G2. With the HSP60 connection to the mitochondria, we hypothesize that APOL1 is imported into the proximal tubule from the basal (serum) and is trafficked to the mitochondria. As a result of APOL1‐associated CKD stress, imported APOL1 loses proper conformation and requires HSP60 for refolding. APOL1 G1 and G2 variants lack the appropriate residues in the HSP60 binding site, resulting in a misfolded conformation and improper signaling. To test the uptake of APOL1 in vitro, HK‐2 cells, which do not express APOL1, were cultured with 10% human serum for a timecourse extending up to two days, harvested and immunoblotted with APOL1 antibody. APOL1 protein levels, but not mRNA levels, were detected after 24 hours of treatment, suggesting an uptake of serum APOL1. The preliminary findings support the hypothesis of APOL1 entering through proximal tubule from the serum and having a possible functional role in the mitochondria involving HSP60.

Support or Funding Information

NIH DK0007470
The Ca2+‐Activated Chloride Channel TMEM16A Colocalizes with CFTR Chloride Channels in Rodent Secretory Airway Epithelial Cells
Authors: Hahn Anne K , Faulhaber Johannes , Srisawang Lalita , Salomon Johanna J , Mall Marcus M , Möhrlen Frank , Frings Stephan
Published: November 2023

There is a variety of animal models available to study cystic fibrosis, a disease caused by mutations in the CFTR gene. However, the applicability of these models is limited as CFTR−/− mice and also CFTR deficient rats fail to recapitulate phenotypic features of the human disease in the lung. One reason may be compensatory effects of Ca2+‐mediated Cl− secretion in airway epithelia. The purpose of this study was to investigate whether the chloride channel TMEM16A accounts for the molecular basis of Ca2+‐activated Cl− conductance in rodent airway epithelia. Furthermore, the spatial expression of the chloride channels, TMEM16A and CFTR, was studied in tissue of the respiratory system of rats and mice.

Rat tracheal epithelial cells were cultured under air‐liquid culture conditions for at least 14 days and subsequently the short circuit current (ISC) was measured in Ussing chambers. For immunohistochemical stainings, respiratory epithelia of the nose, trachea and lungs of mice and rats were co‐stained using anti‐TMEM16A and anti‐CFTR antibodies in combination with markers for different epithelial cell types (e.g. ciliated cells, goblet cells, club cells)

Bioelectrical studies revealed that the UTP‐induced increase in ISC reflecting the Ca2+‐activated Cl− secretion was significantly inhibited by 95% after preincubation with the specific TMEM16A inhibitor Ani9 (ISC values of 0,6 ± 0,5 μA/cm2 (pretreated) vs.12,6 ± 4,3 μA/cm2 (non‐treated)) in rat tracheal epithelial cells. In contrast to human airway epithelia, immunohistochemical stainings in rodents showed a colocalization of TMEM16A and CFTR in secretory epithelial cells throughout the respiratory system. Both channels were not expressed in ciliated cells.

In conclusion, TMEM16A inhibitor Ani9 selectively inhibits UTP‐induced Ca2+‐activated Cl−‐ currents accounting for TMEM16A‐mediated Cl− conductance. The colocalization of TMEM16A and CFTR in secretory cells of rodents suggests a potential regulatory interaction.

Support or Funding Information

Studienstiftung des deutschen Volkes
Temperature Dependence of Delayed Ca2+ Activation of TMEM16F (ANO6) and Its Ca2+ Sensitivity
Authors: Lee Mi‐Ok , Lin Haiyue , Nam Joohyun
Published: November 2023

TMEM16F, also known as ANO6, belongs to a family of putative Ca2+‐activated Cl− channels (CaCCs). In contrast to other classic CaCCs such as TMEM16A (ANO1), the activation of ANO6 requires very high cytosolic Ca2+ concentration ([Ca2+]c > 3 μM) and strong positive membrane potentials. Moreover, even when [Ca2+]c is high, the ANO6 current appears very slowly, requiring >10 min to reach a steady‐state. These properties were confirmed in all three functioning variants (V1, 2, and 5) of ANO6. The low Ca2+‐sensitivity and slow kinetics of ANO6 suggest that it does not function as an ion channel. In the present study, using whole‐cell patch recordings, we found that the three ANO6 isoforms (V1, 2, and 5) transiently transfected into HEK293T cells were activated by lower [Ca2+]c at physiological temperature (37°C). However, V2 and V5 showed significantly higher sensitivity and were activated by 400 nM [Ca2+]c and V1 by 1 μM [Ca2+]c. Furthermore, V2 and V5 showed significantly greater shortening of the latency time of activation than V1; after preparing the whole‐cell configuration, the currents reached a maximum after 543.3 ± 72.48 s, 56.67 ± 9.55 s, and 62.5 ± 4.53 s for V1, V2, and V5, respectively. The temperature‐dependent effect was reversible. In inside‐out patch, all isoforms of ANO6 exhibited higher calcium sensitivity and channel activity at 37°C than at room temperature. V3 showed no current activity in the inside‐out configuration. These results suggest that nearly all isoforms of ANO6 can be activated at low Ca2+ concentration (≤1 μM) when conditioned at physiological temperature, although they showed different kinetics. Our results suggest that ANO6 functions as ion channel in various tissues and cells depending on the variant forms of ANO6.

Support or Funding Information

This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning(NRF‐2015R1C1A1A02037738)
MRP4 Influences cAMP Levels in Ciliary Epithelial cells of the Human Eye
Authors: Pelis Ryan M , Ingraham Leslie , Hotchkiss Adam G
Published: November 2023

The ciliary epithelium is important for aqueous humor inflow and maintenance of intraocular pressure. Treatment of ciliary epithelium with β‐adrenergic agonists leads to increased extracellular cAMP and adenosine and stimulation of aqueous humor inflow. The mechanism by which cAMP gets out ciliary epithelial cells is unknown. The purpose was to determine if the multidrug resistance‐associated protein 4 (MRP4) in ciliary epithelial cells influences intracellular and extracellular cAMP levels. Intracellular and extracellular cAMP levels were monitored in cultured ciliary epithelial cells using a FRET‐based assay. Also examined by RT‐PCR in cultured cells and native human ciliary body was the expression of MRP4, and the expression of receptors and enzymes associated with the cAMP‐adenosine signaling pathway. MRP4 mRNA and protein (immunoblotting) were detected in cultured cells and native human ciliary body. There was a dose‐dependent increase in both intracellular and extracellular cAMP levels following treatment of the cells with isoproterenol (ISO, 0–1 mM). Intracellular cAMP levels were ~10‐fold higher in the presence vs. absence of ISO (1 mM), and extracellular cAMP went from being undetectable to detectable. The β2‐adrenergic receptor was detected in the cultured cells and native ciliary body, but β1‐ and β3‐adrenergic receptors were undetectable. MRP4 mRNA was undetectable by qPCR and RT‐PCR in cultured cells following treatment with siRNA against MRP4. There was no difference in basal intracellular cAMP levels between cells treated with negative control siRNA (control) and MRP4 siRNA. Following treatment with ISO (1 mM) intracellular cAMP levels were 2.7‐fold significantly higher in cells treated with MRP4 siRNA compared to control. There was no difference in extracellular cAMP levels between the two treatments. Also present in both cultured cells and native ciliary body were message for enzymes necessary for conversion of cAMP to adenosine, such as ecto‐5‐nucleotidase (CD73). These data show that MRP4 likely contributes to efflux of cAMP from ciliary epithelial cells where it is then converted to adenosine. The cultured cells should prove useful for further study of the cAMP‐adenosine signaling pathway in the ciliary epithelium of the human eye.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada
Molecular mechanisms mediating the adaptive regulation of human colonic thiamine pyrophosphate (TPP) uptake process
Authors: Srinivasan Padmanabhan , Anandam Kasin Yadunandam , Haidary Habib Ahmad , Said Hamid
Published: November 2023

Thiamin(vitamin B1) is indispensable for normal cellular metabolism due to itsinvolvement in critical metabolic pathways (e.g., energy metabolism, maintenanceof normal cellular redox state, normal mitochondrial structure and function). Human(mammals) cannot synthesize thiamin, and thus, must obtain the vitamin fromexogenous sources, i. e., diet and gut microbiota. A substantial amount of themicrobiota‐generated thiamin exists in the form of TPP. We have previously shown the existence of a specific and high‐affinity carrier‐mediated uptake process for TPP in human colonocytes (Am J Physiol Gastrointest Liver Physiol. 2012; 303: G389‐95), and have recently determined the molecular identity of the system involved (product of the SLC44A4 gene) (J Biol Chem. 2014; 289: 4405‐16). We have also shown that the colonic TPP uptake process is adaptively regulated by the extracellular substrate level, but nothing is known about the molecular mechanism(s) that mediates this regulation. We addressed this issue in the current investigation using the human colonepithelial NCM460 cells as a model. Maintaining these cells in the presence of high levels of TPP (1 mM) for 2 and 9 days was found to lead to a significant(p ≤ 0.01) reduction in the rate of TPP uptake compared to uptake by cells maintained in the absence of added TPP. While no change in the level of expression of the SLC44A4 mRNA was found in cells maintained for 2 days in the presence of high TPP level [suggesting possible involvement of post translation mechanism(s)], a significant (p ≤ 0.01) decrease in level of SLC44A4 mRNA was found in cells maintained for 9 days. We also observed a decrease in activity of the SLC44A4 promoter in cells maintained for 9 days in high TPP level. The latter was associated with clear histone modifications at the SLC44A4 promoter that involve significant decrease in histone H3K4‐trimethylation and H3K9‐acetylation (euchromatin/activating markers)and a significant increase in histone H3K9‐trimethylation and H3K27‐trimethylation (heterochromatin/repressive markers). While maintaining the cells for 9 days in the presence of high TPP level did not affect the level of expression of the two transcription factors, ELF3 and CREB‐1, that drive the activity of the SLC44A4promoter, their binding to the SLC44A4promoter was decreased as shown by Chip‐qPCR analysis. These studies demonstrate, for the first time, that multiple mechanisms are involved in the adaptive regulation of colonic TPP uptake by extracellular substrate level. At early stages, this regulation involves post‐translational mechanism(s), while long‐term adaptive regulation involves transcriptional and epigenetic mechanisms.

Support or Funding Information

Supported by grants from the DVA and the NIH (DK56061 and DK58057).
First identification of mutations in the human SLC5A6 gene associated with brain, immune, bone and intestinal dysfunction
Authors: Subramanian Veedamali , Benke Paul , Constantinescu Alexandru , Said Hamid
Published: November 2023

Biotin (vitamin B7) is indispensable for normal cellular metabolism due to its involvement in a variety of critical metabolic pathways including fatty acid, amino acid and energy metabolism; it also plays a role in regulating cellular level of reactive oxygen species and gene expression, as well as normal immune function/response. Human (mammalian) cells cannot synthesize biotin endogenously; rather they obtain the vitamin across the plasma membrane via a carrier‐mediated uptake process that involves the human sodium‐dependent multivitamin transporter (hSMVT; product of the SLC5A6 gene); this system also transports pantothenic acid and lipoate. We report here, using whole exome sequencing (GeneDx), the first identification of two mutations in the SLC5A6 gene in a young child: R94X [(CGA>TGA) c280 C>T] and R123L [(CGC>CTC), c368 G>T]. Both of these mutations are located in exon 3 of the SLC5A6 gene. The child exhibited many clinical manifestations including failure to thrive, microcephaly, brain changes, cerebral palsy, developmental delay, immunodeficiency, severe gastro‐esophageal reflux, osteoporosis and pathologic bone fractures. After identification of the hSMVT mutations, the child responded favorably to supplemental administration of pharmacological doses of biotin, pantothenic acid and lipoate. Experimental characterization of the identified mutations utilizing human‐derived intestinal HuTu‐80 and brain U87 cell lines, showed impaired functionality (3H‐biotin uptake) of the two identified hSMVT mutants. In addition, our results (using live‐cell confocal imaging) showed poor expression and cytoplasmic localization of the R94X mutant, while the R123L mutant was predominantly retained in the endoplasmic reticulum. This is the first reporting of mutations in the human SLC5A6 gene that lead to defects in hSMVT function and cell biology, and is associated with a host of clinical abnormalities.

Support or Funding Information

Supported by a grant from the DVA, by NIH grants DK58057, DK56057, DK10747, and the Gordon Foundation
Synthetic Biology for Tackling Antimicrobial Resistance
Authors: Lu Timothy
Published: November 2023

Over the last 50 years, exponential increases in our ability to manipulate electrons and engineer electronic systems spawned the information technology revolution. Similarly rapid improvements in technologies for reading and writing DNA are now transforming our capacity to engineer biological systems. Leveraging these technologies, synthetic biology is an emerging discipline for designing biological systems with novel functionalities. This field has opened up new strategies for interrogating, understanding, diagnosing, and treating human diseases. I will discuss two relevant examples where we have: (1) created highly specific antimicrobial agents to detect and combat antibiotic‐resistant bacteria and (2) built new technologies for massively parallel combinatorial genetics in bacteria and human cells that enables the discovery of novel therapeutics for drug‐resistant diseases.
Short‐term exposure to chronic intermittent hypoxia (CIH) to model repetitive hypoxemias of sleep apnea is sufficient to confer protection from brain ischemia
Authors: Toney Glenn M. , Wellford Alexander J. , Andrade Mary Ann
Published: November 2023

Sleep Apnea (SA), a form of sleep disordered breathing, is characterized by repetitive episodes of arterial hypoxemia resulting from interrupted breathing. Often SA results in develop of hypertension, which studies demonstrate results from increased vasoconstriction driven by excessive sympathetic nerve activity. The sympathetic activation response involves recruitment of pre‐sympathetic neurons in the hypothalamic paraventricular nucleus (PVN). Clinical studies report that SA increases the risk of stroke by ~4‐fold. By contrast, pre‐clinical studies in mice suggest that prolonged (~30 days) exposure to CIH is neuroprotective in the middle cerebral artery occlusion (MCAO) model of stroke. Thus available data suggest that neural, glial and/or vascular adaptations to repetitive hypoxia confer protection from brain ischemia. To gain insight into mechanisms of CIH‐induced ischemic protection, effects of short‐term (7 days) exposure to moderate CIH (21% to 10% O2, 10 episodes/h, 8 h/day) on brain tissue infarct volumes were quantified. This was accomplish using a permanent MCAO model as well as a photothrombic lesioning (PTL) model of focal ischemic injury. Results indicate that exposure to CIH for just 7 days reduced the estimated volume of MCAO‐ and hypothalamic PTL‐induced focal ischemic infarcts. In the MCAO model, CIH reduced infarct volume by 31 ± 4 % (SEM, n=5) relative to normoxic controls. CIH more effectively reduced infarct volume in the hypothalamic PTL model by 65 ± 9 % (SEM, n=7). These findings indicate that CIH exposure for only 7 days is sufficient to induce robust protective adaptations. PO2 recordings from multiple brain regions indicate that episodes of CIH cause rapid reductions of tissue O2 availability. However, episodes of brain tissue hypoxia showed little change over consecutive days of CIH exposure, suggesting that CIH does not induce progressive cerebrovascular adaptations that protect brain tissue from experiencing acute hypoxia. Thus findings suggest that adaptations among resident neurons and/or glia promote tissue survival in the face of focal ischemia.

Support or Funding Information

Support: NIH HL088052 & AHA 15GRNT25710176 (GMT)
Dysregulation of Glutathione Synthesis by Enteric Glia During Gastrointestinal Inflammation
Authors: Brown Isola A.M. , Gulbransen Brian D.
Published: November 2023

Enteric glia, a unique class of peripheral neuroglia, are integral in the maintenance of enteric neuronal circuits and normal gastrointestinal (GI) function. In vitro studies have identified the antioxidant reduced glutathione (GSH) as a vital neuroprotective factor secreted by glia (Abdo et. al 2010). However, little is known about the cellular mechanisms regulating GSH production and the role GSH plays during in vivo inflammation. Here, we identified the cellular components involved in GSH synthesis and investigated the effects of in vivo inhibition of GSH synthesis during GI physiology and pathophysiology.

We used immunohistochemistry to localize GSH synthesis proteins and to quantify changes in neuronal density and other cellular markers. We inhibited GSH synthesis in vivo and in vitro with the selective inhibitor Buthionine Sulfoximine (BSO) and induced in vivo inflammation using the 2,4‐dinitro benzene sulfonic acid (DNBS) model of colitis. All experiments used male C57Bl/6 mice, aged 6–8 weeks. Data were analyzed using a Student's t‐test or one/two‐way ANOVA as appropriate.

Glutathione is synthesized in a two‐step process. The first and rate‐limiting enzyme glutamate‐cysteine ligase (GCL) is expressed in enteric glia and neuronal varicosities. The second enzyme, glutathione synthetase, is similarly found in both neuronal and glial cell bodies. Immunoreactivity for both enzymes is upregulated in response to in vivo inflammation (p < 0.005). Inhibition of GSH production with the GCL‐inhibitor BSO is sufficient to drive neurodegeneration both in vivo and in vitro (p = 0.053 and p < 0.01 respectively). Mice drinking water supplemented with BSO were protected from colonic shortening (p = 0.054), inflammatory cell infiltration (p < 0.001) and neurodegeneration during colitis.

Our works highlights a novel role for the endogenous antioxidant GSH and shows that GSH production by enteric glia is an important neuroprotective mechanism during GI (patho)physiology.

Support or Funding Information

F31DK107232 to IB

R01DK103723 to BDG
Mechanisms Involved in the Changes of Synaptic Transmission in the Nucleus Tractus Solitarius of Rats Submitted to Sustained Hypoxia
Authors: Lima‐Silveira Ludmila , Accorsi‐Mendonça Daniela , Bonagamba Leni G.H , Machado Benedito H
Published: November 2023

The sustained hypoxia (SH) is associated with changes in synaptic transmission in the nucleus tractus solitary (NTS) and also with inflammatory processes. In this study, we investigated the effects of anti‐inflammatory treatment on the excitatory synaptic transmission in the NTS neurons and the gene expression of pro‐inflammatory cytokines [interleukin‐1β (IL‐1β), intereleukin‐6 (IL‐6) e tumor necrosis factor‐alfa (TNF‐α)] in this region. Four groups of juvenile wistar rats (100–130g) were evaluated: 1) control (FiO2=20.8%) treated with vehicle (saline + distilled water, i.p), 2) control treated with the anti‐inflammatory Minocycline (30mg/kg, i.p), 3) SH (FiO2=10%) treated with vehicle, 4) SH treated with Minocycline. The experimental protocols were approved by the institutional ethics committee (CEUA 070/2007 and 094/2013). The rats were treated for 3 days and then exposed to SH or normoxia protocol for 24 h. At the end of this period, electrophysiological records of NTS neurons were performed using the whole cell patch‐clamp technique. Genetic analyzes of NTS punches were performed using real‐time qRT‐PCR. The results showed significant changes in the frequency of excitatory postsynaptic spontaneous currents (sEPSCs) in neurons of rats treated with vehicle submitted to SH (2,4±0,3 Hz, n=9) when compared to their control group (0,96±0,2 Hz, n=12; p<0,05). This increase was not observed in neurons of minocycline treated rats submitted to SH (0,7±0,2 Hz, n=9) compared to their control group [(0,7±0,1 Hz, n=10) two‐way ANOVA followed by Bonferroni's post‐test]. A significant increase of the IL‐1β gene expression was observed only in the SH vehicle rats. No changes were observed in relation to cytokines IL‐6 e TNF‐α. These results indicate that the inflammatory process induced by SH, probably due to IL‐1β, participates of the mechanisms related to the increase of excitatory synaptic transmission in NTS neurons.

Support or Funding Information

CAPES, CNPq, FAPESP.
Ablation of Astrocytes in the Paraventricular Nucleus Disrupts the Blood‐Brain Barrier and Increases Blood Pressure in Rats
Authors: Setiadi Anthony , May Clive N , Yao Song T
Published: November 2023

Background

Perivascular astrocytes make direct connections to the cerebral vasculature. This close apposition has been shown to be critical for the regulation of blood‐brain barrier (BBB) integrity and function. Recent studies have shown that the BBB becomes disrupted in hypertensive rats, which allows circulating peptides such as angiotensin II, which is normally excluded from the central nervous system, to access brain areas critical for blood pressure control. To date, the association between astrocytes, the BBB and hypertension is still poorly understood.

Objective

To determine the importance of astrocytes in maintaining BBB integrity and central control of cardiovascular homeostasis.

Methods

Two groups of male Sprague‐Dawley rats (250–400g), anesthetized (pentobarbital, 60 mg/kg i.p.) and conscious freely‐moving, were bilaterally microinjected with sodium fluoroacetate (50 nL; 10 mM), a specific glial toxin, into the paraventricular nucleus of the hypothalamus (PVN) to ablate perivascular astrocytes. Blood pressure and heart rate were continuously recorded throughout. Evans blue dye (4 mL/kg) was then infused intravenously following microinjections to assess BBB integrity. Changes in baroreceptor reflex function were also studied following fluoroacetate administration.

Results

Ablation of PVN astrocytes caused a significant increase in Evans Blue dye extravasation (+16.67 ± 4.9 mean density in arbitrary units, P<0.05) when compared with saline injected controls, indicating a compromised BBB. Furthermore, we found a significant increase in mean arterial pressure in anesthetized (+41.02 ± 5.5 mmHg from baseline, P<0.0001) and conscious rats (+23.82.96 ± 7.8 mmHg from baseline, P<0.05) at 100‐minute and 2‐days following fluoroacetate injection, respectively. However, no changes were seen in the baroreceptor reflex control of heart rate.

Conclusion

Our results show a functional link between astrocytes, BBB disruption and central regulation of cardiovascular function. These findings suggest that a disrupted BBB, via astrocyte dysfunction, may contribute to the pathophysiology of centrally‐induced cardiovascular diseases such as hypertension.
Hypertension is accompanied with over‐activity of immunity
Authors: Shen Xiao (Gregory) Z. , Zhao Tuantuan , Shi Peng , Bernstein Kenneth E.
Published: November 2023

Immune system plays an important role in the pathogenesis of hypertension. To understand whether hypertension affects immunity, we studied antigen presenting cells (APCs) isolated from mice with hypertension induced by angiotensin II or L‐NAME. The APCs derived from hypertensive mice were superior in presenting antigens in vitro. To study antigen presentation in vivo, we immunized sham and AngII treated wild type mice with ovalbumin. Consistently, more OVA‐specific CD8+ T cells were induced in the blood and the spleen of hypertensive mice. Moreover, RIP‐mOVA mice were studied because this transgenic mouse line expressing membrane‐bound OVA in pancreatic islet β cells. RIP‐mOVA mice were made hypertensive with either angiotensin II or L‐NAME. When OVA‐specific OT‐I T cells were i.v. infused into RIP‐mOVA mice, the hypertensive mice had more OT‐I cells infiltration into the pancreas and they developed diabetes more rapidly. These data suggest that hypertension itself is associated with over‐activity of the adaptive immune system.

Support or Funding Information

National Nature Science Foudation of China 31270950 and 81670378
Haemodynamic Mechanisms underlying Essential Hypertension Change Across the Adult Age‐Span: The Anglo‐Cardiff Collaborative Trial
Authors: McEniery Carmel , Yasmin Y , McDonnell Barry , Cockcroft John , Wilkinson Ian
Published: November 2023

Essential hypertension is often viewed and treated as a uniform condition. However, the condition encompasses a number of distinct patterns of blood pressure (BP) elevation such as isolated systolic (ISH), systolic diastolic (SDH) and isolated diastolic hypertension (IDH), which appear to vary in prevalence across the adult age‐span. We hypothesized that different haemodynamic mechanisms account for the age‐related differences in the prevalence of these hypertensive subtypes.

Detailed haemodynamic data including seated (clinic) BP, cardiac output (CO), stroke volume (SV), peripheral vascular resistance (PVR) and aortic pulse wave velocity (aPWV) were available in 5337 individuals (2381 males), aged 18–92 years. All individuals were free of cardiovascular disease and medication. Within each gender, subjects were stratified according to age (<30 years, 30–60 years and >60 years) and hypertensive subtype, on the basis of clinic BP readings.

ISH was the most common hypertensive subtype in males aged <30 years and was characterized by an elevated CO and SV compared with normotensive individuals. In contrast, SDH and IDH were more common in females aged <30 years and associated with elevated PVR and aPWV compared with normotensives. SDH was the most common subtype in males and females aged 30–60 years and accompanied by increased PVR in both genders. ISH was again the most common subtype in individuals aged >60 years. However, in contrast to younger adults, ISH affected both males and females equally (1:1) and was characterized by a marked elevation in aPWV compared with normotensive individuals.

Distinct haemodynamic mechanisms are associated with different hypertensive subtypes in an age‐ and gender‐dependent manner. Detailed physiological profiling and subsequent targeting of therapy towards primary haemodynamic abnormalities could lead to more effective interventions in essential hypertension.
Differential Effect of Slow‐Pressor Angiotensin II on Contractile Responses to Norepinephrine in Arteries with and without Sympathetic Innervation
Authors: Kopf Phillip G , Johnson Alan Kim , Phelps Laura E , Schupbach Chad D , Peuler Jacob D
Published: November 2023

There is evidence that chronically elevated circulating angiotensin II alters the function of the systemic vasculature. In the present study we examined the effect of two weeks of in vivo infusion of a slow‐pressor dose of angiotensin II on vascular responsiveness to exogenous angiotensin II and norepinephrine applied in vitro. As target vessels, we chose to isolate 1) the thoracic aorta, which in the rat is essentially free of sympathetic innervation, 2) a third order mesenteric resistance artery, which like all resistance vessels is densely innervated, and 3) the most proximal portion of the caudal artery, which is densely innervated, but functions in a thermo‐regulatory mode rather than as a resistance vessel. At the completion of the infusion there were no changes in contractile responses to angiotensin II itself in any of the three arterial tissue preparations (intact rings). However, we found that contractile responses to norepinephrine were significantly increased in aortic, but not in mesenteric or caudal arterial rings. For aorta, these increases were accompanied by a significant decrease in the half‐maximally effective concentration of norepinephrine from 48 ± 5 to 31 ± 5 nanomolar in vehicle versus angiotensin II treated rats (n =10 each). Therefore, we propose that long‐term systemic administration of a slow‐pressor dose of angiotensin II can potentially increase expression of alpha‐adrenergic receptors in arterial smooth muscle to increase responsiveness to norepinephrine. However, because it is likely that angiotensin II is acting through central mechanisms to increase arterial vascular sympathetic nerve activity, increased receptor expression may be prevented in densely innervated arterial vessels. Accordingly, our results suggest that angiotensin II‐induced hypertension, at least up to 2 weeks, may not involve increased responsiveness of resistance vessels to either angiotensin II itself or sympathetic nerve activity.

Support or Funding Information

University of Iowa and Midwestern University
Association Between Gender, Age, Body Weight and Hypertension in Nigeria
Authors: Emiloju Opeyemi C. , Chinedu Shalom N. , Onuoha Micheal C. , Iheagwam Franklyn N.
Published: November 2023

Rising incidence of hypertension or high blood pressure (HBP) has been reported amongst Nigerian adult population. According to the American Heart Association, blood pressure of 140 and/or 90 diastolic and above that stays high over time is categorized as HBP. Overweight and obese individuals are said to be at high risk of developing HBP. This study examined the correlation between gender, body weight and HBP amongst workers in a Nigerian University. A total of 727 subjects (280 female; 447 male) within the age bracket of 20–39 (410); 40–59(281); ≥60 (36) participated in this study. The weight, height, systolic and diastolic blood pressure of subjects were measured using standard methods. BMI was calculated as weight per square of height (kg/m2) and the body weight was categorized into underweight, normal weight, overweight and obesity using WHO cut offs. Prevalence of HBP amongst the subjects was 34.8%; it was higher in male subjects (38.26%) compared to the females (29.29%). There was disparity in the prevalence of HBP among the age groups; it was 23.9%, 46.7% and 72.2% respectively for 20–39, 40–59 and ≥60 years. Over 75% of the hypertensive subjects were overweight or obese; prevalence of the body weights amongst the hypertensive subjects was 0.8% (underweight), 24.1% (normal), 33.2% (overweight) and 41.9% (obesity). The study suggests that gender, age and body weight could be important factors in the development of HBP. Further studies involving larger number of subjects are needed to establish the correlation between gender, body weight and HBP in Nigeria.
Genetic Interference with Endothelial PPARg Alters Vascular Responses to Select Renin Angiotensin System (RAS) Peptides
Authors: De Silva Travice , Hu Chunyan , Kinzenbaw Dale , Modrick Mary , Sigmund Curt , Faraci Frank
Published: November 2023

Pharmacological activation of the nuclear receptor peroxisome proliferator‐activated receptor‐gamma (PPARg) protects the vasculature. Much less is known regarding the impact of PPARg under physiological conditions, when driven by endogenous ligands. Recently, we found that endothelial PPARg protects against angiotensin II (Ang II)‐induced vascular dysfunction. Here, we explored that concept further examining whether effects were sex‐dependent along with underlying mechanisms. We studied transgenic mice expressing a human dominant negative mutation in PPARg driven by the endothelial specific vascular cadherin promoter (E‐V290M), using age‐matched, non‐transgenic (non‐Tg) littermates as controls. Acetylcholine (an endothelium‐dependent agonist) produced similar relaxation of carotid arteries from non‐Tg and E‐V290M mice. In contrast, incubation of isolated carotid arteries with Ang II (1 nM) overnight reduced responses to acetylcholine by about 50% in male and female E‐V290M mice but not non‐Tg controls (P<0.05). Endothelial dysfunction in E‐V290M mice was restored to normal by inhibitors of superoxide (tempol), NADPH oxidase (VAS‐2870), Rho kinase (ROCK 1 and ROCK2, Y‐27632), ROCK2 (SLX‐2119), NF‐kB (NEMO‐binding domain peptide), or IL‐6 (neutralizing antibody). Responses in non‐Tg mice were not affected by these inhibitors. We next hypothesized that in addition to vascular effects of Ang II, PPARg may influence components of the angiotensin 1‐7 (Ang 1‐7) arm of the RAS. In the basilar artery, vasodilation to Ang 1‐7 was reduced in E‐V290M mice by more than 50% under baseline conditions (while responses to acetylcholine were minimally affected), an effect that was reversed by Y‐27632. Thus, effects of Ang II on vascular function are augmented by interference with endothelial PPARg through mechanisms that are sex‐independent but involve interrelated oxidant‐inflammatory signaling and Rho kinase (ROCK2). The study provides the first evidence that endothelial PPARg interacts with components of the Ang 1‐7 arm of the RAS. These cell‐specific roles for PPARg have implications for vascular pathophysiology and therapeutic approaches for vascular disease.

Support or Funding Information

This work was supported by the NIH, the Department of Veterans Affairs, and the Fondation Leducq.
Microbial Metabolite, Butyrate, Increases Blood Pressure in Rodent Models of Hypertension
Authors: Yang Tao , Magee Kacy , Malphurs Wendi , Vickroy Thomas , Zubcevic Jasenka
Published: November 2023

Introduction

The emerging link between gut dysbiosis and hypertension is characterized by a significant reduction in butyrate‐producing bacteria in the spontaneously hypertensive rat (SHR) compared with the Wistar Kyoto (WKY) control. Butyrate, a major end product of dietary fiber fermentation by gut microbiota, reportedly produces beneficial effects in multiple dysbiosis‐related diseases. However, butyrate has also been linked with activation of the sympathetic drive, elevated heart rate and release of norepinephrine. Considering the established link between sympathetic drive and hypertension, we investigated the role of butyrate in blood pressure (BP) control in hypertension.

Methods

Male SHR (5 wo) were placed on either the fiber‐rich (butyrolytic) diet (SHR‐BD) or control diet (SHR‐CD) for 13 weeks ad libitum. Fecal/cecal butyrate levels were determined using high performance liquid chromatography (HPLC) throughout the study. Following this, fecal matter transplant (FMT) from both groups was performed separately to male WKY recipients (7 wo) with antibiotic‐depleted endogenous gut bacteria, to create two groups: WKY FMT‐BD and WKY FMT‐CD. Five weeks following FMT, both groups were infused with a subpressor dose of angiotensin II (Ang II; 50ng/kg/min) for three weeks. Separate male SHR (9 wo) were administered with daily injections of either saline or butyrate (500mg/kg/day I.P.) for four weeks. BPs were measured in all groups using tailcuff or telemetry throughout the study.

Results

Elevated cecal butyrate in SHR‐BD (by 2 fold vs. SHR‐CD; P<0.001) was associated with significantly higher SBP (SHR‐CD: 183.3mmHg vs SHR‐BD: 206.6 mmHg; P<0.01). FMT from SHR‐CD and SHR‐BD caused a similar BP increase in the WKY recipients (ΔSBP=11.5 mmHg, P<0.001), However, a subsequent administration of subpressor Ang II further elevated SBP in the WKY FMT‐BD group only (by 7.7 mmHg vs. WKY FMT‐CD; P=0.06). Finally, chronic IP injection of butyrate in the SHR resulted in significant SBP increase compared to saline‐injected SHR (ΔSBP=14.5 mmHg, P<0.05).

Conclusion

SHR gut microbiota is pro‐hypertensive in the WKY. Moreover, subpressor Ang II exacerbates this hypertension in the presence of excess butyrate. Thus, elevated butyrate may interact with Ang II in the pathophysiology of hypertension, indicating the disadvantageous role of butyrate in the context of hypertension.

Support or Funding Information

1. American Heart Association 2. College of Veterinary Medicine, University of Florida
17‐Hydroxyprogesterone caproate improves fetal growth restriction possibly by reducing inflammation and placental cytolytic NK cells in response to placental ischemia during pregnancy
Authors: Amaral Lorena Machado , Elfarra Jamil , Cornelius Denise C , Cunningham Mark W , LaMarca Babbette
Published: November 2023

Preeclampsia (PE), new onset hypertension, is characterized by elevated tumor necrosis factor (TNF‐α), anti‐angiogenic factor soluble fms‐like tyrosine kinase (sFlt‐1), natural killer (NK) cells, oxidative stress and placental ischemia predicted with increased uterine artery resistance (UARI). These contributing factors are likely contributors to the decreased fetal weight during PE pregnancies. Cytolytic NK are an important arm of the immune system killing tumors and infected cells by perforin‐granzyme‐mediated cytolysis which have been shown to be increased in PE women compared to normal pregnancy. Currently, there is no effective treatment for PE except for early delivery, making PE the leading cause for premature births worldwide. Administration of 17‐hydroxyprogesterone caproate (17‐OHPC) is approved for cessation of preterm labor in pregnancies not complicated by PE. The objective of this study was determine whether early administration of 17‐OHPC could improve pregnancy outcomes in response to placental ischemia. To do so, 17‐OHPC (3.32mg/kg) was administered intraperitoneally on gestation day 15 to reduced uterine perfusion pressure (RUPP) rats. UARI was measured using Doppler ultrasound and carotid catheters were inserted on day 18. Blood pressure (MAP), nitric oxide (NO), placental cytolytic NK cells and perforin‐grazyme levels were measured on GD 19. MAP in normal pregnant (NP) rats (n=12) was 94 ± 2, 126 ± 2 in RUPP (n=27) and 111±1 mmHg in RUPP+17‐OHPC (n=15), p<0.05. Pup weight was 2.3±0.09 in NP, 1.9± 0.04 in RUPP rats, which improved to 2.1±0.06 grams in RUPP+17‐OHPC p<0.05. UARI was 0.6±0.01 in NP (n=3), 0.8±0.03 in RUPP rats (n=4), which improved to 0.6±0.04 in RUPP+17‐OHPC (n=5), p<0.05. Total number of placental NK cells was 8.6 ± 3.1 in NP (n=3), 20.2 ±2.4 in RUPP rats (n=6), which decreased to 1.6 ± 0.54 % in RUPP+17‐OHPC (n=5), p<0.05. Activated placental NK cells was 3.8 ± 2.2 in NP (n=3), 11.9±2.01 in RUPP (n=6), which improved to 0.4 ± 0.2 % in RUPP+17‐OHPC (n=5), p<0.05. Perforin‐granzymes were 0.04±0.001, 0.004±0.001 in RUPP (n=4), which improved to 0.02±0.003, 0.0008±0.0001 pg/ug protein in RUPP+17‐OHPC (n=6), p <0.05. Plasma NOx was 22.9±1.6 in NP (n=9), 10.82±2.3 in RUPP rats (n=13) which improved to 25.5±5.2 μM in RUPP+17‐OHPC (n=5), p<0.05. Administration of 17‐OHPC improves UARI, NO bioavailability, activated cytolytic NK cells, perforin‐grazymes, pup weight, and hypertension in response to placental ischemia indicating the potential benefit of 17‐OHPC to improve NK cell modulated pathogenicity and pregnancy outcomes associated with PE.

Support or Funding Information

NIH grants RO1HD067541‐06, T32HL105324
Leptin Role in Regulating Ovariectomized Induced Hypertension
Authors: Barnes Maria J. , Hackman Sophia M
Published: November 2023

Hypertension is less prevalent in pre‐menopausal women compared to males of a similar age. Once women become menopausal, the incidence of hypertension increases significantly when compared to men. Though it has been established that estrogen plays a protective role against the development of hypertension in pre‐menopausal women, it is not clear what the underlying mechanisms might be. Most menopausal women experience a significant increase in body weight in the form of adipose tissue. Increases in adiposity is accompanied by increased levels of the “satiety hormone”, leptin, that also have cardiovascular regulatory properties. Thus, utilizing a menopausal rat model, we sought to test the hypothesis that leptin plays a role in increasing blood pressure and heart rate. Ovariectomized (OVX) adult female rats (i.e., 4‐week post‐surgery) were used in these experiments. OVX rats weighed more, had higher leptin and lower estrogen levels when compared to control females. Blood pressure and heart rate were significantly higher in OVX rats as well. When leptin was administered intracerebroventricularly into the control animals we observed a small decrease in blood pressure. However, administering the same dose in OVX rats resulted in an increase in blood pressure and heart rate. Taken together, these data suggest that estrogen dampens leptin's influence on blood pressure homeostasis and with the loss of estrogen, the influence of leptin on cardiovascular function is not inhibited.

Support or Funding Information

Iowa Osteopathic Educational Research Foundation