bluuebunny/crossref_metadata_2025 huggingface dataset:
Fibroblast growth factor 21 is critically involved in alcohol‐induced hepatic lipid metabolism and adipose tissue lipolysis in mice
Authors: Zhao Cuiqing , Liu Liming , Yang Ying , McClain Craig , Feng Wenke
Published: November 2023

Purpose

Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator produced primarily in the liver that regulates hepatic fat metabolism in various models of metabolic syndromes. Alcoholic liver disease (ALD) is characterized by hepatic lipid accumulation. Here, we sought to determine the role of FGF21 in mice exposed to alcohol and to discern underlying mechanisms.

Methods

Male FGF21 knockout (KO) and control (WT) mice were fed either the Lieber DeCarli diet containing 5% alcohol (AF) or an isocaloric diet (PF). For the chronic alcohol exposure, mice were fed for 4 weeks. For the chronic‐binge alcohol exposure, mice were fed for 12 days, followed by a single dose of alcohol (5g/kg) or isocaloric maltose dextrin treatment by gavage. Mice were sacrificed 6 hours later. One group of AF mice were administered with recombinant human FGF21 (rhFGF21) for the last 5 days. Liver steatosis and inflammation and epididymal white adipose tissue (eWAT) lipolysis were investigated.

Results

Alcohol exposure induced plasma FGF21 elevation. FGF21 knockout exacerbated chronic alcohol‐induced hepatic steatosis and liver injury, which was associated with increased activation of genes involved in lipogenesis mediated by SREBP1c and decreased expression of genes involved in fatty acid (FA) β‐oxidation mediated by PGC1α. Chronic alcohol exposure induced a moderate adipose tissue lipolysis in eWAT. rhFGF21 administration reduced alcohol‐induced hepatic steatosis and inflammation in WT mice. Surprisingly, in the chronic‐binge model, alcohol consumption‐induced fatty liver is blunted in the KO mice. Chronic‐binge alcohol exposure‐induced in situ hepatic lipogenesis and FA β‐oxidation were comparable to that in chronic alcohol‐exposed mice. However, FGF21 deficiency markedly reduced chronic‐binge alcohol exposure‐induced eWAT lipolysis, which may contribute to hepatic FA uptake. Further analysis showed that in the KO mice alcohol induced significant elevation in systemic catecholamine, which is known to promote adipose lipolysis. rhFGF21 administration increased alcohol‐ induced fat loss in parallel with an increase of circulating norepinephrine concentration in KO mice.

Conclusion

Our results demonstrated that the role of FGF21 in ALD is alcohol exposure model dependent. Lack of FGF21 exacerbates chronic alcohol exposure‐induced fatty liver, and this exaggeration is overridden in chronic‐binge alcohol exposed mice through a reduction of adipose lipolysis mediated by systemic catecholamine release involving sympathetic nervous system activation. Pharmacologic strategies targeting hepatic FGF21 elevation and reduction in adipose tissue lipolysis are warranted.

Support or Funding Information

Supported by the NIH and Veterans Administration
Sulfation of Environmental Estrogens by Human Cytosolic Sulfotransferase (SULT) 1C4
Authors: Guidry Amber L , Runge‐Morris Melissa , Falany Charles N
Published: November 2023

Sulfation plays an important role in the metabolism of drugs and hormones, the bioactivation of carcinogens, and the detoxification of xenobiotics. Human cytosolic sulfotransferase (SULT) 1C4, a Phase II conjugating enzyme, catalyzes the transfer of the sulfonate group from 3′‐phosphoadensosine 5′‐phosphosulfate (PAPS) to the hydroxyl or amine group of the substrate. SULT1C4 expression has been reported to be highest in fetal lung and fetal kidney, with lower expression in the fetal heart and adult kidney, ovary, and spinal cord. SULT1C4's expression suggests a role in detoxication defenses across the maternal‐fetal barrier and integration of hormonal and metabolic signals that are generated during organ development. Unlike many of the other cytosolic SULTs, the role of SULT1C4 in endogenous metabolism and physiology remains poorly understood; however, previous studies have shown that SULT1C4 can sulfate environmental estrogen‐like compounds, such as bisphenol A (BPA). Numerous studies have shown that exposure to environmental estrogens can have harmful effects on fetal development. The objective of this study is to characterize the sulfation of environmental estrogens by SULT1C4 using molecular modeling and kinetic analysis. Native SULT1C4 was bacterially expressed with the pKK233‐2 bacterial expression vector and purified using affinity chromatography (Ni‐NTA) and DEAE‐ion exchange chromatography. To identify environmental estrogens sulfated by SULT1C4, we performed a combination approach, including modeling, pharmacophore analysis, and kinetics. Pharmacophore analysis helps to initially screen potential substrates by fitting possible substrates into a defined catalytic pocket; lead compounds were tested for sulfation by SULT1C4 using a [35S]PAPS thin layer chromatography assay. Daidzein and BPA appear to be better substrates for SULT1C4 than genistein. In silico substrate docking into the SULT1C4 active site did not identify daidzein as a substrate suggesting that the current model for SULT1C4 (PDB 2GWH) may not be predictive of substrate binding, or daidzein binds only to the unliganded (open) enzyme. Inhibition assays using [14C]‐1‐naphthol identified apigenin as an inhibitor of SULT1C4. Our data suggests that SULT1C4 sulfates selected environmental estrogen‐like compounds, including phytoestrogens and industrial chemicals, although some of these compounds may be non‐catalytic competitive inhibitors. Further characterization of SULT1C4's substrate reactivity is needed to gain a better understanding of its role in physiology and metabolism.

Support or Funding Information

This work was supported by NIH ES022606 and GM038953.
Cardiac calsequestrin and ageing
Authors: Neumann Joachim , Fahrion Carola , Fabian Stephanie , Gergs Ulrich
Published: November 2023

Cardiac calsequestrin (CSQ2) is the main Ca2+ binding protein of the mammalian heart. Patients with deletion of CSQ2 are susceptible to lethal catecholamine‐induced arrhythmias but in the absence of systolic heart failure. To further characterize the role of CSQ2 for regulation of cardiac contractility we generated CSQ2‐null (CSQ2−/−) mice by targeted deletion of the first exon (heterozygous: CSQ2+/−; wild type: CSQ2+/+). In the targeted CSQ2 allele, the coding region of exon 1 is replaced by a loxP site leading to a nonsense mutation. Chimeric mice were crossed into a C57BL/6 background to get CSQ2+/− mice for further breeding. Successful targeting of the CSQ2 allele was confirmed by Western blotting. CSQ2−/− mice were viable. Ventricular function as assessed by Langendorff perfusion in mice six months of age was already altered: basal contractility in CSQ2−/− mice was higher (possibly a compensatory mechanism) than in CSQ2+/− and CSQ2+/+ mice. Additionally, at six months gross morphology of the heart as well as absolute and relative heart weight were not different in CSQ2−/− mice compared to CSQ2+/+ or CSQ2+/−. However, in 20–22 months old mice we measured systolic heart failure (possibly as a result of breakdown of compensatory mechanisms) as assessed by decreases in ejection fraction (in echocardiography using a Vevo system and isoflurane anesthesia) in CSQ2−/− mice (29 ± 6.1%, n= 4) compared to six month old CSQ2−/− mice and to 20–22 months old CSQ2+/− mice (68 ± 6.4% n=5) and CSQ2+/+ (65 % ± 6.8%, n=4, p< 0.05). In summary, we present evidence that deletion of CSQ2 can lead to cardiac failure upon ageing, which might be relevant for human carriers of CSQ2 mutations.
Caveolin‐3 Regulation of Cardiac Contractile Function is Facilitated by Enhanced Mitochondrial Efficiency
Authors: Busija Anna R. , Schilling Jan M. , Roth David M. , Insel Paul A. , Patel Hemal H.
Published: November 2023

Caveolins (Cavs) are scaffolding proteins integral to caveolae, membrane lipid raft domains that compartmentalize numerous proteins and signaling molecules, including β‐adrenergic receptors (β‐ARs). Mice with cardiac myocyte (CM)‐specific overexpression of Cav‐3 (Cav‐3 OE) have more caveolae in CMs and are protected from ischemia‐reperfusion injury. Here, we tested the hypothesis that increases in Cav‐3 expression in CMs may alter β‐AR response, especially since data from our laboratory have shown mitochondria from Cav‐3 OE hearts are resistant to calcium loading and generate fewer reactive oxygen species. We reasoned that Cav‐3 OE, by compartmentation of interdependent signaling components of the β‐AR response in close approximation to mitochondria, may change responses to β‐AR activation. Cav‐3 OE hearts treated with the β‐AR agonist isoproterenol (Iso) have augmented inotropy (+dP/dt) and lusitropy (−dP/dt) compared to transgene negative hearts. CMs isolated from Cav‐3 OE mice have a 2‐fold increase in maximal Iso‐promoted cAMP production. However, isolated Cav‐3 OE CMs incubated with Iso show only a small increase in mitochondrial respiration. Thus, increased expression of Cav‐3 enhances Iso‐mediated positive inotropy without a proportional increase in mitochondrial respiration. We conclude that increased expression of Cav‐3 enhances cardiac function by enhancing responsivity of the heart and increasing mitochondrial oxygen efficiency in response to βAR stimuli, thus identifying an increase in Cav‐3 as a novel means to increase cardiac function in a manner that may not require increased oxygen consumption.

Support or Funding Information

NIH HL107200, AHA 14PRE20460372.
Role Of The NLRP3/Inflammasome On Aldosterone‐Induced Vascular Damage
Authors: do Nascimento Thiago Bruder , Ferreira Nathanne , Zanotto Camila , Campos Eduardo , Ramalho Fernanda , Orlandin Isabela P. , Olivon Vânia Claudia , Neves Karla Bianca , Lopes Rhéure A.M. , Silva Carlos Alberto , Carlos Daniela , Mestriner Fabiola , Alves‐Filho José Carlos , de Cássia Tostes Rita
Published: November 2023

Aldosterone (Aldo), a steroid with mineralocorticoid activity, is closely associated with vascular inflammation, remodeling and dysfunction. NLRP3/inflammasome, a member of cytoplasmic NOD‐like receptors, plays important role on inflammatory processes. We postulated that NLRP3/inflammasome mediates aldosterone‐induced vascular damage. To test this hypothesis we analyzed vascular structure and function and inflammatory profile of wild‐type (WT), NLRP3 knockout (NLRP3−/−) and Caspase‐1 knockout (Casp‐1−/−) mice treated with vehicle or Aldo (600 ug/kg/day for 14 days, via osmotic mini‐pump) while receiving 1% saline to drink. Aldo infusion did not change the final mean arterial pressure (WT: 101,9 ± 4,8 vs Aldo: 107,4 ± 5,1), but increased plasma levels of IL‐1β (WT: 588,0 ± 103 vs Aldo: 2333,9 ± 658,8* pg/mL, *P<0.05), as well as cleaved caspase‐1 and mature IL‐1β in the mesentery arteries, determined by ELISA and western blot, respectively, in WT mice. Apocynin (antioxidant agent) treatment prevented Aldo‐induced effects. NLRP3−/−and Casp‐1−/− mice were protected against Aldo‐induced vascular changes, i.e. they did not exhibit hypercontractility to phenylephrine [maximal effects (Emax) (WT: 75,2 ± 9,6 vs Aldo: 155,1 ± 22,1* % of KCl‐induced contractility, *P<0.05)] or reduced ACh‐induced relaxation upon Aldo treatment maximal effects [(Emax) (WT: 87,7 ± 2,1 vs Aldo: 34,2 ± 3,3*, % of relaxation, *P<0.05)]. Aldo infusion increased media:lumen ratio and media cross‐sectional area of resistance arteries, augmented RNA expression for inflammatory markers [(ICAM‐1 and VCAM‐1) 3 and 5‐folds, respectively] and the number of adhered macrophages in aortic segments. These Aldo‐induced changes were blunted in NLRP3−/−and Casp‐1−/− mice. To elucidate the role of NLRP3 in the vasculature, we performed bone marrow transplantation (BMT) from WT to NLRP3−/− mice (WTàNLRP3−/−), BMT chimeric mice were protected from Aldo‐induced vascular dysfunction and remodeling. All together these data suggest that NLRP3/inflammasome activation in the vasculature is oxidative stress‐dependent, and plays a crucial role on Aldo‐induced vascular dysfunction and remodeling.

Support or Funding Information

Financial Support: Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) e Coordenação de Aperfeiçoamento de Ensino Superior 88887.092495/2015‐00 (CAPES), Brazil.
The role of cytochrome P450 1B1 and its associated mid‐chain hydroxyeicosatetraenoic acids metabolites in the development of cellular hypertrophy induced by isoproterenol
Authors: El‐Kadi Ayman O.S. , Maayah Zaid H.
Published: November 2023

Heart failure is the leading cause of death, with a prevalence of over 23 million worldwide. The lifetime risk of developing heart failure is one in five. Numerous experimental studies have demonstrated the role cytochrome P450 1B1 (CYP1B1) and its associated mid‐chain hydroxyeicosatetraenoic acids (mid‐chain HETEs) metabolites in the pathogenesis of heart failure and cardiac hypertrophy. However, the ability of isoproterenol (ISO) to induce cardiac hypertrophy through mid‐chain HETEs has not been investigated yet. Therefore, we hypothesized that ISO induced cardiac hypertrophy through the induction of CYP1B1 and its associated mid‐chain HETEs metabolites. To test our hypothesis, the human ventricular cardiomyocytes, RL‐14 cells, were treated with ISO in the presence and absence of tetramethoxystilbene (TMS), a selective CYP1B1 inhibitor. Thereafter, the cellular hypertrophy markers, cell volume and mid‐chain HETEs metabolites were determined using real‐time polymerase chain reaction, phase contrast imaging and liquid chromatography‐electron spray ionization‐mass spectrometry, respectively. Our results showed that ISO induced cellular hypertrophy in RL‐14 cells as evidenced by the significant induction of β‐myocin heavy chain/α‐myocin heavy chain (β‐MHC/α‐MHC) and cell volume. Interestingly, ISO‐induced cellular hypertrophy was associated with a proportional increase in the protein expression of CYP1B1 and the formation of mid‐chain HETEs metabolites. The direct evidence for the involvement of CYP1B1 in the ISO‐induced cellular hypertrophy was supported by the ability of TMS and CYP1B1 siRNA to significantly inhibit the ISO‐mediated the induction of β‐MHC/α‐MHC and the increase cell volume. Mechanistically, the protective effect of TMS against ISO‐induced cellular hypertrophy was mediated through the inhibition of NF‐κB signaling pathway. In conclusion, our study provides the first evidence that the inhibition of CYP1B1 and hence mid‐chain HETEs attenuate ISO‐induced cellular hypertrophy.

Support or Funding Information

This work was supported by a grant from the CIHR to A.O.S.E.
Deficiency of Prostaglandin E2 Receptor Subtype 4 Causes Dysregulation of Cholesterol Homeostasis in Mice
Authors: YING FAN , CAI Yin , TANG Eva HoiChing
Published: November 2023

Mice lacking prostaglandin E2 receptor subtype 4 (EP4), one of the four receptors identified for prostaglandin E2 developed spontaneous hypercholesterolemia. The present study investigated the cause of the elevated plasma cholesterol in EP4 knockout mice. 12–15 weeks old normal diet fed EP4 knockout mice show a 48% increase in total cholesterol as compared to their wildtype littermates. The proportion of plasma cholesterol present in very low density lipoprotein (VLDL)/low density lipoprotein (LDL) increased by 84% and that in high density lipoprotein (HDL) increased by 15.6%. The increase in plasma cholesterol was independent of changes in intestinal cholesterol absorption, as there were no changes in fecal neutral sterol excretion and in intestinal ATP‐binding cassette (ABC) A1 and G5 protein expression between EP4 wildtype and knockout mice, while hepatic ABCA1 was significantly increased in the latter – suggesting that the deficiency of EP4 increases cholesterol efflux from the liver. The absence of EP4 reduced phosphorylation of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMG‐CoA reductase) and in so doing increased HMG‐CoA reductase activity, the rate‐limiting enzyme for cholesterol synthesis. Furthermore, deficiency of EP4 reduced hepatic mRNA (by 23%) and protein expression (by 36.7%) of low density lipoprotein receptor (LDLR), suggesting that there is impaired cholesterol clearance by the liver. Such conclusion was further reinforced by studies where EP4 knockdown in human hepatoma cell lines reduced cell surface LDLR protein, impaired LDL uptake and reduced intracellular cholesterol. Lastly, EP4 deficiency decreased the catabolism of cholesterol to bile acids. Cholesterol 7α‐hydroxylase (CYP7A1) is the rate‐limiting step in the classic pathway of bile acid biosynthesis, and its mRNA expression levels were decreased by 57.8% in livers of EP4 deficient mice as compared to those in wildtype mice. While, the expression of sterol 27‐hydroxylase (CYP27A1) which initiates the alternate or acidic pathway of bile acid biosynthesis was unchanged. Indeed, EP4 knockout mice exhibited higher plasma but lowered hepatic levels of total bile acids. In summary, the present study shows that the hypercholesterolemia in EP4 deficient mice is mediated by multiple mechanisms including increased hepatic cholesterol biosynthesis, increased cholesterol efflux from the liver, impaired cholesterol clearance by the liver, reduced conversion and/or elimination of cholesterol into bile acids. The lack of EP4 in mice leads to an incapacity to maintain normal plasma cholesterol levels, suggesting a loss of coordinate regulation for cholesterol homeostasis, identifying a new metabolic dimension in the physiological role played by endogenous EP4.

Support or Funding Information

This work is supported by The Research Grant Council Early Career Scheme (ECS) Project code: 27101314.
Activation of TGF‐β/Smad signaling pathway by high cholesterol diet: possible inhibition by rutin compound
Authors: Alrejaie Salim S. , AlSharari Shakir , Hafez M.
Published: November 2023

Background

High‐cholesterol diet (HCD) increases the oxidative stress in liver resulting in generation of reactive oxygen species (ROS). ROS may activate transforming growth factor beta (TGF‐β) and Mothers Against decapentaplegic Homolog (Smad)signaling pathway leading to hepatotoxicity. Rutin is a natural flavonoid (vitamin p), which possesses an antioxidant activity with protective potential. The aim of the present study was to investigate the potential alleviating effects of rutin against hyper‐cholesterolemia‐induced TGF‐β/Smad signaling pathway activation and hepatotoxicity in rat.

Methods

Male Wistar rats were divided into four groups: G‐I control (Rat chow), G‐II Rutin (0.2% in rat chow), G‐III HCD (1% cholesterol and 0.5% cholic acid in rat chow) and G‐IV rutin (0.2%) + HCD. Animals received freshly prepared experimental diets for 6 consecutive weeks. In hepatic tissue, gene expressions of TGF‐β, Smad‐2 and Smad‐4 were measured using real time PCR.

Results

In HCD group, the gene expression levels of TGF‐β, Smad2 and Smad4 significantly increased by8‐, 8.5‐ and 5.3‐folds expressions, compared to the control group. On the other hand, administration of rutin in combination with HCD induced a significant repair of the HCD‐induced alteration in the gene expression of TGF‐β, Smad2 and Smad4 compared to the HCD group expression levels.

Conclusion

Our findings revealed that TGF‐β/Smad signaling pathway is involved in the hepatotoxicity induced by HCD and rutin inhibits hepatotoxicity via suppressing this pathway. Therefore, rutin might be considered as a protective agent for hepatotoxicity.

Support or Funding Information

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research project no NFG‐14‐02‐13
The Integration of Mechanobiology and Biomechanics at Primary Cilia
Authors: Jacobs Christopher Rae , Nguyen An
Published: November 2023

Cellular mechanosensation is critical in diseases responsible for enormous human suffering including atherosclerosis, osteoarthritis, cancer, and osteoporosis. Nonetheless, very little is understood about the molecular mechanisms of mechanotransduction outside of a small number of specialized sensory cells. Primary cilia are solitary linear cellular extensions that extend from the surface of virtually all cells. For decades, the biologic function of these enigmatic structures was elusive, however, recent evidence suggests an emerging picture in which the primary cilium functions as a complex nexus where both physical and chemical extracellular signals are sensed and responses coordinated.

In our laboratory we have shown that primary cilia act as mechanical sensors in bone and that conditional deletion of primary cilia lead to mechanosensing defects. Recently, we developed a novel combined experimental/modeling approach to determine the mechanical properties of primary cilia. We found a wide variety of previously unreported deformation modes including smooth bending and rigid‐body rotations. This suggests that the mechanics of both the cilium shaft and basal anchorage are important to understanding deflection patterns. Interestingly, both the cilium itself and its anchorage to the microtubule cytoskeleton alter their structure in response to physical loading, suggesting structural adaptation or “remodeling”. We have also developed novel molecular biology tools to elucidate the details of mechanically activated ciliary signaling pathways. For example, we have created a cilia‐directed biosensor that has allowed us to distinguish intraciliary from intracellular calcium signaling. We have also developed a method for distinguishing the roles of the cytoplasmic and ciliary pools of proteins that are found in both compartments. In summary, primary cilia are non‐linear, richly varied, mechanical structures (biomechanics) as well as structurally adaptive (mechanobiology). Simultaneously they are a biochemical microdomain where signaling events are catalyzed, enhanced, and integrated.

Support or Funding Information

Supported by NIH AR45989 and AR54156 and New York State Stem Cell Grant N089‐210
Caspase‐1 P20 Fragment, a Marker for Inflammasome Activation, is Elevated in Serum of Patients during Myocardial Infarction
Authors: Morgan Andrew , Liang Bruce , Chen Kai , Na Doohwan , Gold Courtney , Azrin Michael , Lee JuYong , Schumacher Jayne , Yue Lixia , Aguila Hector Leonardo , Shapiro Linda , Tate Janet , Protas Matthew
Published: November 2023

Aims

Increased inflammation contributes to the development of myocardial infarction (MI) and impacts adversely infarct healing. Caspase‐1 is an end effector of inflammasome activation and is an important mediator of systemic inflammation. Caspase‐1 is cleaved to produce a 20 kDa peptide upon activation. We tested the hypothesis that the 20kDA peptide is elevated in patients during and after acute myocardial infarction.

Methods and Results

Between 12/10/2007 and 8/10/2015 complete data for caspase‐1, troponin, CK, and CKMB during the acute event and the post‐MI period were obtained in 75 consecutive subjects with type 1 acute myocardial infarction. The average age was 61.8±14 years. Using a solid phase enzyme‐linked immunosorbent assay, the 20 kDa peptide was detected in serum during ST elevation and non‐ST elevation MI and its peak level(166.2±11.2 pg/mL) was higher than the non‐peak level(96.3±6.2 pm/mL, p=<0.0001) during the acute phase. The peak level was higher than the level after the acute MI (termed post‐MI, 115.4±8.5 pg/mL, p=<0.0001), and both levels were higher than that of healthy subjects (79.19±5.3 pg/mL). The peak level was not correlated with peak cardiac cTnI or peak CK‐MB.

Conclusions

Activated caspase‐1 20 kDa peptide rises and falls during acute MI. The post‐MI level remains elevated and may represent persistent inflammation. The data provided evidence for inflammasome activation during acute MI in humans. The elevated circulating 20 kDa peptide may have implications as biomarker for cardiac injury and healing.
mTORC1 Signaling Mediates Cytoprotective Effects of H2S in Cerebral Circulation
Authors: Liu Jianxiong Dianna , Basuroy Shyamali , Leffler Charles W , Parfenova Helena
Published: November 2023

Cortical astrocytes are key functional components of the neurovascular unit. Our previous study in newborn pigs has demonstrated that astrocyte functions in cerebral blood flow regulation are extensively impaired following neonatal seizures. A cytoprotective gaseous mediator, H2S, enzymatically produced by cortical astrocytes or released by a donor compound, NaHS, prevents deleterious effects of seizures on astrocyte‐mediated cerebral vascular functions. However, the mechanism of H2S cerebroprotection remains elusive. Serine/threonine protein kinase mTOR is a critical component of the cell survival mechanism during stress conditions. Our novel finding suggests that rapamycin‐sensitive mTOR complex 1 (mTORC1) is highly expressed in cortical astrocytes from newborn pigs. We tested the hypothesis that mTORC1 is a key signaling component in H2S‐mediated astrocyte protection against cerebral vascular stress caused by neonatal seizures. The role of mTORC1 in the newborn pig cerebral circulation was tested using rapamycin (0.5 mg/kg ip). Rapamycin reduced mTOR phosphorylation at Ser2448 in cortical astrocytes, indicating effective inhibition of astrocytic mTOR activity. In control intact pigs, rapamycin had no effects on systemic (mean arterial blood pressure, heart rate, core body temperature) or cerebral vascular parameters (diameter of pial arterioles, the major resistance vessels in the brain). Epileptic seizures were induced by bicuculline (3 mg/kg ip) in control saline‐treated piglets or in piglets pretreated with NaHS (3 mg/kg ip), without or with co‐administered rapamycin. Astrocyte‐mediated cerebral vascular functions were tested during delayed postictal period (48 h after seizures) by responses of pial arterioles to astrocyte‐dependent vasodilators (ADP, glutamate, and ionotropic glutamate receptor agonist quisqualate) by live microscopy using the cranial window technique. In control postictal pigs, all astrocyte‐dependent pial arteriolar responses were greatly reduced, whereas the astrocyte‐independent vascular smooth muscle responses (isoproterenol, sodium nitroprusside) remained intact. Systemically administered NaHS had remarkably strong cerebroprotective effects and completely prevented the loss of astrocyte‐dependent vascular responses in postictal pigs. In contrast, in piglets with rapamycin‐inhibited mTORC1 activity, NaHS failed to rescue the neonatal brain from the seizure‐induced loss of astrocyte‐dependent cerebral vascular responses. We conclude that mTORC1 signaling mediates the protective effects of H2S in the cerebral circulation and is critical in preventing the loss of astrocyte functions in cerebral blood flow regulation caused by neonatal seizures.

Support or Funding Information

The study was supported by NIH/NHLBI/NINDS.
Doxorubicin‐Induced Chronic Upregulation of Transforming Growth Factor‐beta
Authors: Mancilla Trevi Ann , Andrews Thomas , Aune Gregory
Published: November 2023

As a result of their treatment, childhood cancer survivors face an increased risk of acute and chronic heart failure. Epidemiological studies have shown the anticancer drug doxorubicin, while effective, to be cardiotoxic. Patients suffering from chronic doxorubicin cardiotoxicity exhibit interstitial fibrosis, one component of heart failure. This project aims to elucidate the mechanism of fibrosis in doxorubicin‐induced heart failure by examining changes in the transforming growth factor‐beta (TGF‐β) signaling pathway after exposure to doxorubicin.

Our novel model involves treating 2 week old C57BL/6J mice with five weekly intraperitoneal injections of 3 mg/kg of doxorubicin. Cardiac tissue was collected one week after the final injection and at 80 weeks of age to assess early and long‐term recovery points. Data previously gathered by our lab show increased interstitial fibrosis in doxorubicin‐treated animals, even after exposure ceases. In vitro studies show a direct effect of doxorubicin on cardiac fibroblasts, inducing collagen production at the transcriptional level.

Left ventricular TGF‐β levels increased in an age‐dependent and treatment‐dependent manner, as shown by immunohistochemistry. Cells positively‐stained for TGF‐β were more abundant in doxorubicin‐treated animals at the 7 week and 80 week time points compared to control mice (p=0.03 and p<0.001, respectively). Additionally, positively stained cells were located in the interstitium, indicating that the positive cells were cardiac fibroblasts. Preliminary data indicates an increase in matrix metalloproteinase‐2 (MMP‐2) in the 7 week doxorubicin‐treated mice. As a mediator of collagen degradation, MMP‐2 is an indicator of cardiac remodeling.

Doxorubicin treatment causes sustained molecular changes evidenced by the short‐term and long‐term increases in the signaling cytokine TGF‐β in the murine left ventricle. Early changes in collagen and MMP‐2 indicate a cardiac remodeling phenotype in our model. This project shows that doxorubicin initiates changes to components of fibrosis, possibly driven by activation of the TGF‐β signaling pathway.
Lysyl Oxidase Inhibition Alleviates Fibrosis and Dysfunction in Rodents with Established Cardiac Disease
Authors: Ninh Van Kim , El Hajj Elia C , El Hajj Milad C , Mouton Alan J , Gardner Jason D
Published: November 2023

Heart failure is the end result of many cardiovascular disorders, including myocardial infarction, hypertension, and diabetes. Regardless of etiology, a key mechanism of heart failure is adverse extracellular matrix (ECM) remodeling. Lysyl oxidase (LOX) is a collagen cross‐linking enzyme that is elevated in failing human hearts, and is associated with cardiac fibrosis. Our goal for this study was to determine if inhibition of LOX in rodents with established cardiac disease would provide protection against further adverse cardiac remodeling and dysfunction. The aortocaval fistula rat surgical model of volume overload (VO) was used. Eight weeks after surgery, both sham‐operated and VO rats were treated with a LOX inhibitor, beta‐aminopropionitrile (BAPN; 100 mg/kg/d). Treatment was administered for six weeks using an osmotic minipump inserted into the peritoneal cavity.

Echocardiography was used to assess progressive alterations in cardiac remodeling and function. At 14 weeks, left ventricular (LV) catheterization was used to assess alterations in contractility, stiffness, and other indices of cardiac function. Using LV homogenates and fixed sections, interstitial myocardial collagen (immunohistochemistry), and protein expression of collagens I and III (Western Blot) were measured. Collagen cross‐linking was assessed using pyridinoline (Pyd) assay.

VO induced significant LV hypertrophy (116% increase vs sham), diastolic LV wall stress (363% increase vs sham), fibrosis (172%, 77% and 22% increase in interstitial myocardial collagen, collagen I and collagen III, respectively, vs sham; p<0.05), and cardiac dysfunction (17% decrease in ejection fraction vs sham). LOX inhibition partially attenuated VO‐induced increases in LV hypertrophy (23% decrease vs VO; p<0.05). Inhibiting LOX also completely attenuated VO‐induced increases in interstitial myocardial collagen, and protein expression of collagens I and III (180%, 76% and 22% decrease, respectively, vs VO; p<0.05). LOX inhibition significantly attenuated increases in cardiac wall stress (33.5% decrease vs VO) and conferred cardioprotective effects as indicated by improved fractional shortening (19% increase vs untreated VO; p<0.05). LOX inhibition also significantly attenuated VO‐induced decreases in contractility, assessed using end systolic pressure‐volume catheterization and preload recruited stroke work (70% and 30% increase, respectively, vs VO; p<0.05). Overall, our data demonstrate the cardioprotective effects of LOX inhibition on the volume overload stressed heart, and its potential to slow or even prevent the transition to heart failure.

Support or Funding Information

Supported by the NIH/NCRR #P20RR016456 and the American Heart Association Greater Southeast Affiliate #11GRNT7700002 (jdg).
Chloroquine Attenuates Klotho Gene Deficiency‐induced Arterial Stiffening and Hypertension by Suppression of Autophagy
Authors: Chen Kai , Sun Zhongjie
Published: November 2023

Background & Hypothesis

Hapodeficiency of the antiaging gene Klotho causes arterial stiffening and hypertension through unknown mechanisms. Our recent study showed that autophagy was upregulated in the aortas of Klotho mutant heterozygous (+/−) mice. We hypothesized that inhibition of autophagy by chloroquine (an anti‐malaria drug) attenuates Klotho deficiency‐induced arterial stiffening and hypertension.

Methods & Results

Klotho mutant heterozygous (+/−) mice and age‐ and sex‐matched wild‐type (WT) mice were used (20–23 months). Pulse wave velocity (PWV), a direct measure of arterial stiffness, and blood pressure (BP) were increased significantly in KL(+/−) mice. Interestingly, inhibition of autophagy by chloroquine reversed Klotho deficiency‐induced increases in PWV and BP within 2 weeks of treatment. The autophagy level, as measured by LC3I/LC3II and autophagy flux, was increased in aortas of KL(+/−) mice. Chloroquine abolished the upregulation of autophagy in Klotho (+/−) mice, indicating effective inhibition of autophagy. Klotho deficiency‐induced arterial stiffness was associated with accumulation of stiffer collagen and degeneration of compliant elastin fibers. In addition, the activity and expression of MMP2, MMP9, and the expression of TGF‐β1, TGF‐β3, RUNX2 and ALP were increased in aortas of KL(+/−) mice, which were attenuated by chloroquine. Scleraxis, a transcription factor in the regulation of collagen synthesis in arterial cells, was increased in aortas of KL(+/−) mice. The upregulation of scleraxis by Klotho deficiency was eliminated by inhibition of autophagy, which was further confirmed by inhibition of autophagy through siRNA knockdown of Beclin‐1 in mouse vascular aortic smooth muscle cells. Therefore, induction of autophagy is essential for Klotho deficiency–induced upregulation of scleraxis.

Conclusion

Klotho gene deficiency causes arterial stiffening and hypertension through upregulation of scleraxis in an autophagy‐dependent manner.

Support or Funding Information

Supported by NIH R01 HL118558, DK093403, HL105302 and HL102074.
Circadian Rhythm of Pulse Pressure are decreased in Peroxisome Proliferator Activated Receptor‐α Knockout Mice during Angiotensin II Hypertension
Authors: Stukes Ian T , Reece Takisha , Lee Dexter L
Published: November 2023

Circadian rhythm of pulse pressure is considered an index of cardiovascular disease. An elevated pulse pressure is commonly associated with increased stiffness of the aorta due to high blood pressure. Activation of PPAR‐a has been shown to decrease hypertension; however, the role of PPAR‐a on pulse pressure during Angiotensin II hypertension is not well understood. We tested the hypothesis that the absence of PPAR‐α would decrease changes in the circadian rhythm of pulse pressure during Angiotension II (Ang II) hypertension. Male (10 – 12 weeks old) PPAR‐alpha knockout (KO) mice and their wild‐type (WT) control were infused with Ang II (400 ng/kg/min) for 12 days. Day and night systolic, diastolic, pulse and mean arterial pressures, along with heart rate and locomotor activity was collected 19‐hrs/day. The average change in day to night pulse pressure was 1 ± .05 and 3 ± .1 mmHg in Ang II treated KO and WT mice, respectively. No differences in day to night heart rate were observed between KO (30 ± 3 bpm) and WT (30 ± 5 bpm). Day to night changes in locomotor activity, systolic, diastolic and mean arterial pressures were similar between both groups. TBARS were increased in KO + Ang II (15 ± 2 mM) vs WT + Ang II (11 ± 3 mM). Nitrite/Nitrate was decreased in KO + Ang II (1.0 ± 0.1 nM) vs WT + Ang II (1.5 ± 0.5 nM). Interleukin 17 was increased in KO + Ang II (1.5 ± 0.3 ng/mL) vs WT + Ang II (1.0 ± 0.2 ng/mL). Our results suggest that PPAR‐alpha activation increases the circadian rhythm of pulse pressure by decreasing oxidative stress, and IL‐17 concentrations while increasing nitrite/nitrate concentrations during Ang II hypertension.

Support or Funding Information

5K01HL092593‐05
Relationship between basilar artery vasomotion and blood pressure in normotensive and hypertensive rats
Authors: Tomiak‐Baquero Ana Maria , von Linstow Roloff Eva , Kasparov Sergey , Paton Julian F.R.
Published: November 2023

Our group has previously hypothesised (Cates et al., 2012) that hypoperfusion of the brainstem is a contributing factor to the increased blood pressure (BP) in spontaneously hypertensive rats (SHR) in accordance with Cushing's mechanism. Artery vasomotion is a mechanism believed to increase tissue perfusion and oxygenation. In this study, we have assessed whether the basic characteristics of vasomotion in the basilar artery are present in the SHR in vivo and if there are differences between the SHR and normotensive Wistar rats. To our knowledge this is the first time vasomotion and vasoactivity of the basilar artery has been investigated in SHR and Wistar rat with the dura intact, thereby preserving physiological conditions. In addition it is the first time, to our knowledge, that SHR basilar vasomotion is studied in vivo. Under isofluorane anaesthesia, Wistar (n=3) and SHR (n=2) were kept at a constant temperature (37°C) and ventilated mechanically keeping end tidal CO2 around 4.5%. Arterial BP was monitored and a femoral vein cannulated for drug delivery. The ventral brainstem was exposed and the dura thinned to allow visualisation of the basilar artery from the vertebral bifurcation to the anterior inferior cerebellar arteries. Phenylephrine (20–50 μg/min) and sodium nitroprusside (0.6–4.2 μg/min) were injected i.v. to produce stepwise increases and decreases in blood pressure, respectively. In Wistar rats vasomotion occurred between 70 and 170 mmHg and confirms previous data (Fiji et al., 1990). In contrast, our pilot data suggests a higher pressure threshold (>90 mmHg) is needed before vasomotion is initiated in the SHR. We suggest that this may impede arterial perfusion and tissue oxygenation at lower blood pressures in the SHR compared to Wistar rats. These results support our notion of a hypoperfused brainstem in the hypertensive state.

Support or Funding Information

This project was funded by the BHF.
The Anti‐Inflammatory Cytokine IL‐19 Reduces mRNA‐Stability Protein HuR Function in Human Vascular Smooth Muscle Cells
Authors: Herman Allison , Haines Dale , Autieri Michael V.
Published: November 2023

Vascular diseases such as atherosclerosis and restenosis are often exacerbated by associated inflammation. Vascular smooth muscle cells (VSMCs) respond to injury and are capable of producing pro‐inflammatory cytokines which amplify the inflammatory response. Identification of compounds which attenuate this response is an important therapeutic goal. Presently, little is known about the potential for endogenously expressed anti‐inflammatory cytokines to attenuate inflammation and have vascular protective effects. Interleuken‐19 (IL‐19) is a newly identified member of the IL‐10 anti‐inflammatory family. IL‐19 expression can be induced in VSMCs by pro‐inflammatory cytokines and is capable of reducing the stability and abundance of pro‐inflammatory mRNA transcripts. HuR is an mRNA stability protein which functions to stabilize pro‐inflammatory transcripts by shuttling from the nucleus to the cytoplasm where it binds to AU‐rich elements within the 3′ UTR of transcripts. Nuclear to cytoplasmic shuttling is essential for HuR activity. We previously determined that treatment of VSMCs with IL‐19 transiently reduces HuR abundance in the cytoplasm, by a presently unknown mechanism. In the present study we show that overexpression of HuR significantly increases VSMC proliferation. We hypothesize that IL‐19 inhibits HuR translocation to the cytoplasm by altering chaperone protein function, abundance, or both. To test this hypothesis, VSMCs were transfected with epitope‐tagged HuR expression plasmids, followed by treatment with TNFα alone, IL‐19, or both, to determine if any HuR‐interacting proteins are differentially regulated by inflammation or IL‐19 to alter HuR activity. Protein extracts were prepared from each treatment group and HuR was immunoprecipitated and proteomic analysis (MASS SPEC) was performed to identify HuR‐interacting proteins in each of these conditions. Numerous novel protein interactions with HuR are altered by the different inflammatory treatment conditions. The roles of many of these novel interacting proteins involve RNA‐binding and post‐transcriptional regulation based on the literature. Inflammation‐modulated differential HuR protein‐ protein interactions provide a framework showing mRNA stability can be modified by HuR chaperone proteins VSMCs, which is a novel direction of study for inflammation. The proteins identified could lead to a better understanding of the mechanisms associated with vascular inflammatory diseases such as atherosclerosis and provide targets for new therapeutic agents.

Support or Funding Information

This work was supported by grants HL115575 and HL117724 from the National Heart Lung, and Blood Institute of the National Institutes of Health, and Grant 13GRNT1685003 from the American Heart Association to MVA
The role of PKC‐B in the BKCa functionality of vascular smooth muscle of diabetic rats
Authors: Espinosa‐Tanguma Ricardo , Álvarez‐Méndez Juan Antonio , Hernández‐García Ana Patricia , Algara‐Suárez Paola
Published: November 2023

Diabetes mellitus (DM) is a disease characterized by high blood glucose concentrations. Cardiovascular disorders associated with DM are the leading cause of high morbidity and mortality of this disease, where endothelial tissue as well as smooth muscle is damaged. For example, the contractility of the smooth muscle cells is increased partly due to a decrease in the activity of the calcium‐dependent high conductance potassium channels (BKCa), which along with other potassium conductance maintain membrane potential and vascular tone.

Protein kinase C (PKC) is involved in signaling pathways that promote muscle contraction. Christiandimitropoulou (2002) reported that in the vascular smooth muscle of diabetic rats the outwardly currents flowing through the BKCa channels are decreased. Likewise it was also reported that PKC can phosphorylate some residues of the carboxyl terminal of the alpha subunit of said channel. Data from our laboratories show that the inhibition of the classic isoforms of the PKC is associated with a decrease in the contraction of the mesenteric artery of diabetic rats, which suggests that the beta isoform may be involved in the vascular damage observed in DM.

Objectives

To study the role of the PKC‐B on BKCa channels functionality and the aortic vascular contractility of diabetic rats induced by streptozotocine.

Methods

Tension and western blot experiments were performed on aortic rings.

Results

the NS11021, a BKCa channel activator, significantly relaxes the rings of diabetic rats on a lesser scale than those of the control group, the PKC‐B LY333531 inhibitor does not modify the contractility induced by phenylephrine in normal rings but importantly relaxes those from diabetic rings. The effect of NS11021 is preserved even in the presence of LY333531. Western blot experiments suggest that smooth muscle from diabetic rats is less phosphorylated, indicating that BKCa activity is reduced compared with those from control rats.

Conclusions

vascular smooth muscle of diabetic rats show greater contractility and this may be due to a diminished activity of the BKCa channels.

Support or Funding Information

Supported by C14‐FAI‐04‐33.33 to PAS, Conacyt 223350‐M to RET. Conacyt scholarship to JAM (295659)
Clustering and combining pattern of metabolic syndrome risk factors and its association with arterial stiffness
Authors: Vicente Wanda Rafaela , Rodrigues Sara , Dutra‐Marques Akothirene CB , Carvalho Jefferson C , Jordao Camila P , Toschi‐Dias Edgar , Hussid Maria Fernanda , Costa‐Hong Valeria , Bortolotto Luiz A , Cepeda Felipe X , Trombetta Ivani Credidio
Published: November 2023

Introduction

Metabolic syndrome (MetS) is associated with structural and functional vascular abnormalities that can lead to increased arterial stiffness, which predicts cardiovascular events and increases mortality. However, little is known regarding the implications of risk factor clustering in the arterial stiffness.

Objective

In MetS, determine the clustering and the combining pattern of risk factors and its association with arterial stiffness.

Methods

We selected recently diagnosed patients with MetS (n=47, 51 ± 6 y) according to ATP III criteria. A control group (C, n=17, 50 ± 4 years) was also studied. Arterial stiffness was evaluated by pulse wave velocity (PWV) in carotid‐femoral segment. The patients were categorized and analyzed according MetS risk factors clustering (3, 4 and 5 factors). We performed correlation tests and multiple linear regressions to determine predictor variables with PWV and independent variables of sex, age, body mass index, and MetS risk factors.

Results

Patients with MetS had increased PWV compared to C (8.8 ± 0.7, 9.8 ± 1.4 m/s, p=0.01). In multivariate analysis, the variables that remained as predictor of PWV were age (β=0.35, p= 0.01) and triglycerides (β=0.29, p= 0.03). There was a progressive increase of PWV to increase the number of risk factors. Furthermore, the PWV compared to C group was similar for cluster of 3 risk factors, whereas were higher for cluster of 4 and 5 risk factors (8.7 m/s [7.8 to 10.0], 9.4 m/s [7.7to 11.7], 9.8 m/s [7.3 to 13.4] and 10 m/s [9.3 to 13.4]).

Conclusion

The number of risk factors amplifies the arterial stiffness. The presence of alteration in the triglycerides worsening the stiffness of large vessels, emphasizing the importance in addressing this risk factor for MetS patients.

Support or Funding Information

This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP# 2011/17533‐6 ) and, in part, by Fundação Zerbini. Sara Rodrigues was supported by FAPESP# 2013/15323‐0, Edgar Toschi‐Dias was supported by FAPESP# 2013/07651‐7, Felipe X Cepeda was supported by FAPESP# 2015/03274‐0.
Molecular Mechanisms of the Inhibitory Effects of Viscolin on Overgrowth of Smooth Muscle Cells and Neointimal Hyperplasia
Authors: Wu Chiehhsi
Published: November 2023

Percutaneous coronary angioplasty (PTCA) together with stent deposition has been regarded as the most useful approach for treating myocardial infarction. Recent research studies indicated that stent placement leads to mechanical injury and induces substantial local inflammation. The pathophysiological process includes intimal hyperplasia, extracellular matrix (ECM) remodeling, acute inflammatory cell infiltration. Overproduction of reactive oxygen species (ROS) in vascular smooth muscle cells (VSMCs) may stimulate the migration and proliferation of VSMCs leading to neointimal thickening and restenosis. Because viscolin has been known to decrease ROS production in human neutrophils, we aimed to explore if viscolin can inhibit neointimal hyperplasia (NIH) and its molecular mechanisms. The inhibitory effects of viscolin on neointimal formation were then studied in balloon injured rat model. Viscolin or vehicle were delivered to rats by intraperitoneal injection for two weeks every day after balloon injury. Morphological analysis using H & E staining at day 14 indicated that viscolin prevented intimal hyperplasia in rat thoracic aorta as compared to vehicle control. Our results also demonstrated that viscolin significant reduced matrix metalloproteinases ‐2 (MMP‐2) expression in A7r5 cells by western blotting and zymography assay. Our current data suggest that viscolin could be a potential candidate to prevent restenosis after angioplasty by inhibiting cell proliferation and migration.