bluuebunny/crossref_metadata_2025 huggingface dataset:
Administration of the thromboxane receptor antagonist vapiprost in a mouse model of DSS‐induced colitis
Authors: Harris Norman R. , Carter Patsy R.
Published: June 2021

Thromboxane has been implicated in the constriction of intestinal submucosal arterioles in mice rendered colitic with dextran sodium sulfate (DSS). In the present study, C57BL/6 mice given 40 kD DSS were administered one of three doses of the thromboxane receptor antagonist vapiprost: 0.3, 0.5, and 1.0 mg/kg/day, or no vapiprost as a control. DSS and vapiprost were given in drinking water (5% w/v for DSS) for a period of 6 days, concluding with an assessment of a disease activity index comprising stool consistency, rectal bleeding, and weight loss. The presence of loose stools decreased by 30–40% with all doses of vapiprost. Rectal bleeding decreased by ~25% with the lower dose of vapiprost, and by ~40% with the two higher doses. However, vapiprost was unable to prevent DSS‐induced weight loss. The overall disease activity index (0–4 scale) decreased from 2.9 ± 0.1 (no vapiprost) to 2.3 ± 0.3 (p<0.05) with the higher vapiprost doses of 0.5 and 1.0 mg/kg/day. In summary, vapiprost attenuates the disease activity index of DSS‐induced colitis in mice, primarily by decreasing the presence of loose stools and rectal bleeding. Supported by CCFA and NIH (P01DK043785).
In chronic colitis exacerbation of altered motility induced by the bacterial peptide formyl‐Methionyl‐Leucyl‐Phenylalanine (fMLP) is associated with dysregulation of the immune system
Authors: Cruz Myrella L , Alvarado Zellinette , Coppola Domenico , Antonia Scott J , Appleyard Caroline B
Published: June 2021

The pathophysiological role of bacterial peptides in the symptoms associated with colitis is unclear.

Aim:

To investigate the role of fMLP in chronic colitis.

Methods:

Colitis was induced in rats by administration of trinitrobenzene sulfonic acid (TNBS; 30mg in 50% ETOH ic). Chronic relapsed inflammation was induced 6 weeks later by TNBS (5mg/kg iv), with or without the bacterial peptide (1.0mM in 6% DMSO iv) at 24 hour time intervals over three days. The colons were assessed for macroscopic and microscopic damage, and immunohistochemistry. Tissue cytokine levels were measured by ELISA and real‐time RT‐PCR. Segments of longitudinal and circular smooth muscle from proximal and distal colon were mounted in tissue baths for isometric recording in response to Ach.

Results:

Administration of fMLP exacerbated both macroscopic and microscopic damage (p<0.05). Cellular infiltration and increased numbers of T‐cells and dendritic cells were found. These animals had more diarrhea and increased muscle tension in response to Ach. Levels of IFNγ and TNFα increased after administration of fMLP (p<0.05), while IL‐10 levels were decreased (p<0.05), with little change in TGFβ or IL‐18.

Conclusions:

Components of the intestinal microflora may drive a dysregulation of the immune system and influence the contractile responses of the colon in chronic colitis contributing to the symptoms observed in patients. NCRR/RCMI 2G12RR03050
Glucagon induces the protein expression of rat hepatocyte aquaporin‐8.
Authors: soria leandro raúl , gradilone sergio alejandro , larocca maria cecilia , marinelli raúl alberto
Published: June 2021

Glucagon stimulates the vesicle trafficking of aquaporin‐8 (AQP8) water channels to the canalicular membrane of rat hepatocytes, a mechanism thought to be involved in glucagon‐induced bile secretion. Our aim was to study whether glucagon is also able to stimulate the gene expression of hepatocyte AQP8. Glucagon was administered to rats at 0.2 mg/100g body wt ip for 8, 16, and 36 h. By immunoblotting, we found that 34 kDa AQP8 was increased by 79% and 107% in total liver membranes at 16 and 36 h. Hepatocyte AQP9 protein expression remained unaltered. AQP8 mRNA expression, assessed by real time PCR, was not modified over time. Immunoblotting studies in primary cultured rat hepatocytes showed that glucagon increased the total amount of AQP8 (e.g. 80% with 1μM glucagon) and confocal immunofluorescence showed a predominant AQP8 cell surface localization. The RNA polymerase II inhibitor actinomycin D did not prevent glucagon effect supporting the nontranscriptional upregulation of hepatocyte AQP8. Glucagon effect on AQP8 was mimicked by dibutyryl cAMP and supressed by the protein kinase A (PKA) inhibitors, H‐89 or PKI. In conclusion, our data suggest that glucagon induces the posttranscriptional gene expression of hepatocyte AQP8 via a cAMP‐dependent PKA pathway, a process likely relevant to the long‐term modulation of bile secretion. Supported by Grant PICT 31670 from ANPCyT
Smad7 Attenuate Inducible Collagen Production in Hepatic Stellate Cells involving the regulation of P21 and P27
Authors: ZHANG GUO , TAN NING , LIANG LIEXING , ZHANG FA‐CAN , WANG TIANCAI
Published: June 2021

An inhibitory Smad7 emerges as a potential therapeutic candidate to intervene the hepatic fibrosis. Given that Smad7 functions through antagonizing the transforming growth factor (TGF)‐beta1 signaling, Smad7 over‐expression might enhance the proliferation of Hepatic Stellate Cells (HSCs). However, our recent monitor show that Smad7 surprisingly promoted apoptosis in these cells. Here we report that such inhibitory machinery of Smad7 during the TGFbeta1‐derived process is coupled to the activity of cyclin‐dependent kinase inhibitors p21 and p27. Initially, Primary isolated HSCs were transiently transfected with an adenoviruses encoding Smad7 gene. Real‐time PCR and western blot analysis was confirmed that the overexpression of Smad7 substantially attenuated TGFbeta1 induced collagen I mRNA and protein levels in HSCs. In parallel, flow cytometric monitoring was recorded that Smad7 gene transfer dramatically enhanced but not abolished the apoptosis in these cells subjected to TGFbeta1. Interestingly, Smad7 induced apoptosis in HSCs was found to be both p21 and p27 linkage by using a specific small interfering RNA technique. In the present of TGFbeta1 was found to stimulate an obvious elevation in p21 protein and mildly increase in p27. However, it was found that Smad7 significantly induced p27 whereas caused a substantial decrease in p21 levels in these cells. Furthermore, silencing of p27 by specific siRNA was found to synergy with the inhibitory effect of Smad7 on the inducible collagen production in these cells. Collectively, our finds indicate the important roles of p21 and p27 in the inhibitory Smad7 working pattern in HSCs in response to TGFbeta1 and thereby provide a novel intracellular basis for associated drug development in the future. (Supported by Guangxi NSF Grants 0135035)
Alterations in Toll‐like Receptor Regulation in the Liver from Horses with Laminitis
Authors: McGeachy Matthew , Bell Amy , Keowen Michael L , Peiro Juliana , Stokes Ashley M.
Published: June 2021

Laminitis can occur secondary to an ischemic‐reperfusion injury of the GI tract. When the normal flora of the GI or pH is disrupted, stimulators of inflammation can be released into the GI bloodstream. The next point of organ contact is the liver via the portal blood stream potentially leading to increased expression of inflammatory mediators, such as Toll‐like receptors (TLR)‐2 and ‐4.

Liver samples of normal (n=18) and horses with naturally‐acquired (n=5) and experimentally‐induced (n=7) laminitis were collected and TaqMan RT‐PCR was used to quantify TLR‐2 and ‐4. Beta‐Gus was used as the housekeeping gene and standard curves were utilized. Data is presented as delta‐delta CT.

Equine TLR‐2 and ‐4 are upregulated in liver collected from laminitic compared to normal. Upregulation of TLR‐2 in laminitic horses is over 3‐fold higher than normal. Upregulation of TLR‐4 was only a minor increase. However, horses with experimentally‐induced laminitis (48 hours after induction) had the greatest increase compared to chronic cases having a 6‐fold TLR‐4 increase.

Identification of inflammatory mediators may aid in the unraveling of equine laminitis, helping horses such as Barbaro. Also, pinpointing an earlier point in this cataclysmic reaction will help in the development of gene specific therapeutics as this technology is developed and utilized. Funded by Louisiana State University Equine Health Studies Program.
Temporal and dose‐dependent effects of hepcidin in cultured macrophages and intestinal epithelial cells
Authors: Sharp Paul , Chung Bomee , Chaston Timothy , Srai Surjit Kaila
Published: June 2021

Hepcidin regulates iron homeostasis by inhibiting the release of iron from enterocytes and RE macrophages. To achieve this effect, it is proposed that hepcidin binds to the ferroportin transporter, triggering its internalization and degradation. In the present study, we have examined the effects of hepcidin in two human cell lines; Caco‐2 intestinal epithelial cells and THP‐1 macrophages. In THP‐1 cells there was a rapid (within 4h) decrease in ferroportin protein expression following exposure to 1μM hepcidin (control 1.1 ± 0.05 a.u.; +hepcidin 0.5 ± 0.06 a.u., P<0.001). The inhibitory effect of hepcidin on THP‐1 ferroportin levels was dose‐dependent with an IC50 of approximately 300nM hepcidin. In contrast, there was no effect of hepcidin on ferroportin expression in Caco‐2 cells, even when the hepcidin concentration was elevated to 3μM (control 1.0 ± 0.28 a.u.; +hepcidin 1.4 ± 0.26 a.u. P>0.1). These data support the hypothesis that iron‐recycling macrophages are more sensitive than enterocytes to changes in hepcidin levels, and is fully consistent with the paramount importance of macrophages in maintaining the supply of iron for erythropoiesis.

This work was funded by the Biotechnology and Biological Sciences Research Council.
Refeeding‐induced thyrotropin (TSH) secretion: A possible role for PYY3–36?
Authors: Costa‐e‐Sousa Ricardo Henrique , Oliveira Karen Jesus , Almeida Norma Aparecida , Souza Luana L , Cedraz‐Mercez Pedro , Cabanelas Adriana , Pazos‐Moura Carmen Cabanelas
Published: June 2021

Low levels of PYY3–36, TSH and thyroid hormones are adaptative mechanisms to fasting state. Serum TSH increased after PYY3–36 peripheral administration in fasted rats. Since PYY3–36 is secreted by intestinal cells after a meal, it could be involved in refeeding‐induced TSH secretion. Thus, we aimed to investigate how PYY3–36 stimulates TSH secretion and its role in TSH secretion after refeeding. Pituitary glands of fasted rats showed no change in TSH secretion when incubated during 2h in presence of PYY3–36 at different concentrations (10−10, 10−8 and 10−6 M). Serum TSH decreased 15 minutes after PYY3–36 injection (0.1nmol/rat) into the 3rd ventricle of 72h fasted rats (2.02±0.29 vs 1.03±0.1 ng/mL, P<0.01). Reduced dose (0.01nmol) did not modify serum TSH (1.67±0.20 vs 1.95±0.16 ng/mL; control vs PYY3–36). Anyway, if PYY3–36 has a role in refeeding‐induced TSH secretion, TSH level should increase quickly after refeeding. While 1h refeeding did not modify serum TSH (0.98±0.18 ng/mL), 3h increased it, relatively to fasted group (2.99±0.40 vs 1.15±0.23 ng/mL, P<0.001). Therefore, it is possible that PYY3‐36 may play a role in refeeding‐induced TSH increase. However, PYY3–36 does not act directly at the pituitary to modulate TSH secretion of fasted rats. In addition, data do not support the hypothesis that PYY3–36 stimulates TSH secretion by direct hypothalamic action.

CNPq, FAPERJ.
Central fatigue induced by intracerebroventricular infusion of AT1‐receptor blocker is influenced by serotonin content in preoptic area and hypothalamus
Authors: Leite Laura Hora Rios , Soares Danusa Dias , Marubayashi Umeko , Coimbra Cândido Celso
Published: June 2021

We previously showed that central angiotensinergic transmission plays an important role on thermoregulation and metabolic adjustments during exercise by increasing heat dissipation and modulating heat production. To asses the effect of central angiotensin AT1 receptor blockade by losartan (Los) on serotonin content in preoptic area (POA) and hypothalamus (HP), Wistar rats were submitted to exercise until fatigue after intracerebroventricular (icv) injection of 2 μL of Los (60 nmol, n=6) or 0.15 M NaCl (Sal, n=6). Time to fatigue (TTF) and workload (W) were analyzed. At fatigue, animals were decapitated, brains were removed and POA and HP were dissected and frozen in dry ice. Serotonin (5‐HT) and 5‐hydroxyindoleacetic acid (5‐HIAA) were measured by HPLC. Los animals showed a 35% and 32% decrease in TTF and W, respectively, when compared to Sal‐treated rats. These animals showed higher levels of 5‐HT and 5‐HIAA in POA (5‐HT: 0.4±0.05 ng/mg, Los vs 0.09±0.02 ng/mg, Sal; 5‐HIAA: 2.6±0.5 ng/mg, Los vs 0.8±0.2 ng/mg, Sal, p< 0.05) and HP (5‐HT: 0.2±0.02 ng/mg, Los vs 0.06±0.03 ng/mg, Sal; 5‐HIAA: 2.4±0.6 ng/mg, Los vs 1.2±0.4 ng/mg, Sal, p< 0.05). An inverse correlation between W and 5‐HT content in the POA and HP was also found (POA: r= 0.67; HP: r= 0.63, p< 0.05). Our data suggest that central fatigue induced by AT1 receptor blockade is related to an increase in 5‐HT content in the POA and HP.

Supported by: CNPq, FAPEMIG and CAPES.
Development of a Functional Schwann cell Phenotype from Adult Porcine Bone Marrow
Authors: Rutten Michael , Janes Michael Ann , Wang Lian , Chang Ivy , Bazar Ann , Gregory Kenton
Published: June 2021

Adult bone marrow cells (BMC) are a potential resource for use in regenerative medicine such as in the production of Schwann cells for the repair of damaged nerves. However, many reports of BMC‐derived Schwann cells have not shown a functional phenotype. The objective of this study was to induce BMC to differentiate into a cell type with both phenotypic and functional characteristics Schwann cells. Adult porcine bone marrow was collected, enriched by Histopaque density gradient centrifugation, and differentiated in Neurobasal media. After eight days in culture we found that BMC expressed the Schwann cell markers S‐100, O4, GFAP, and were positive for cholesterol using FluoroMyelin. These same cells had low p75 receptor expression but neuregulin (1–25nM) increased p75 levels by 24–48hrs. The BMC‐derived Schwann cells produced an ATP increase in intracellular Ca2+ [Ca2+]i, with agonist potency being UTP = ATP>ADP> AMP = adenosine. The agonists α,β,‐methylene‐ATP had little effect on [Ca2+]i while suramin blocked the ATP‐induced [Ca2+]i suggesting the presence of an ATP purinergic P2Y2 G‐protein coupled receptor. The staining for FluoroMyelin and the ATP‐induced [Ca2+]i changes were all attenuated in cells passaged >5 times in culture. Using an in situ culture assay we found that Quantum‐dot labeled BMC rapidly adhered and differentiated into Schwann cells on damaged nerves. Our studies indicate that adult BMC can be induced to form a functional Schwann cell phenotype that could provide a valuable source of cells for use in nerve regeneration studies.
Differentiation of human circulating fibrocytes into chondrocytes and osteoblasts
Authors: Choi Young Hun , Burdick Marie D. , Strieter Robert M.
Published: June 2021

Fibrocytes are a distinct population of blood‐borne progenitor cells with fibroblast‐like features. It has also been proposed that fibrocytes have multipotency of mesenchymal stem cells. Recently Kurt Hong et al. showed fibrocytes differentiated into adipocytes and fibroblasts (Hong et al. 2007. J. Biol. Chem., 282: 22910–20). We further investigated whether fibrocytes can differentiate into chondrocytes and/or osteoblasts. Under the existence of 10 ng/ml TGF‐beta3, fibrocytes differentiated into chondrocytes, proved by Safranin‐O staining for type II collagen. Von Kossa calcium staining showed that the incubation of fibrocytes with osteogenic medium, containing dexamethasone, ascorbate, and beta‐glycerophosphate, resulted in differentiation of fibrocytes into osteoblasts. In conclusion, circulating fibrocytes have multipotency of adult stem cells, and could be useful for regenerative therapy.

Supported by NIH Grant HL‐66027.
Changes of oxidative stress and antioxidant enzymes during replicative senescence of human mesenchymal stem cells
Authors: Chen Chien‐Tsun , Ho Jennifer H , Cho Hui‐Tzu , Lee Oscar K , Wei Yau‐Huei
Published: June 2021

Mesenchymal stem cells (MSCs) possess good proliferation capabilities. However, the alteration of physiological properties of MSCs during in vitro culture remains elusive. We examined the changes of oxidative stress levels and mitochondria in over‐confluence culture‐induced replicative senescence of MSCs. The results showed that human umbilical cord blood‐derived MSCs (UMSCs) and bone marrow‐derived MSCs (BMSCs) reached replicative senescence at the 40th and 38th population doublings (PD), respectively. Intracellular ROS levels (hydrogen peroxide and superoxide) were increased progressively during replicative senescence and the levels were higher in BMSCs. Moreover, viability assay showed that UMSCs were more resistant to exogenous ROS than BMSCs, and such abilities were declined during senescence. Intracellular ATP levels and mitochondrial mass were increased, and the activities of superoxide dismutase‐2 and catalase were decreased in both cells during senescence. Taken together, the ROS level was increased and antioxidant activities decreased in MSCs during replicative senescence, which occurred in conjunction with functional changes in the mitochondria. The results have offered a new explanation for the reduced tissue repair capacities during aging besides reduction in stem cell number and the antioxidant activities should be taken into consideration when choosing the sources of MSCs.
Arginine magic: Arginine kills tumor cells when presented in saline
Authors: Shukla Jyoti , Thakur V.S. , Poduval T.B.
Published: June 2021

There has been no anti‐cancer formulation so far, which is sufficiently effective for the treatment of cancer using an endogenous molecule. Various scientists have attempted to describe the complex role of arginine in cancer biology and treatment. Nobody has yet looked at the non‐substrate actions of arginine, to act as a Biological Response Modifier through its chemical structure. Arginine containing peptides have been used as delivery vectors to carry payloads of drugs. The results indicate that guanidinium group is critical for the translocation process. Based on the above, we hypothesized that pharmacological doses of L‐arginine (LA) when delivered in saline could interact with tumour cells more efficiently.

Various tumor cell lines derived from mouse and human when incubated with LA, D‐arginine (DA) in pharmacological doses in PBS, caused the death of tumor cells, as assessed by the membrane integrity test, trypan dye exclusion test and MTT assay. There was complete destruction of cell structure (Fig.1). DA had better anticancer ability as it could inhibit the tumor growth at lower concentration. The anticancer property of LA is not accompanied by any toxicity to the red blood cells or NIH 3T3 cells. L‐lysine and agmatine did not offer any antitumor activity. Arginine administered in PBS could also cure the mice of the tumor transplanted into the peritoneum (Fig. 2). These findings provide a previously unrecognized beneficial effect of guanidinium group containing compounds in killing the tumor cells and thus provide new potential approaches for therapeutic development of arginine in tumor therapy.
Aquaporin‐4 expression is altered around the lateral ventricle in correlation with beta‐amyloid plaque expression in a transgenic mouse model of Alzheimer‘s Disease
Authors: Moredock Kaitlin Marie , Cirrito John R. , Holtzman David M. , Krane Carissa Marie
Published: June 2021

Accumulation of beta amyloid (Aβ) is central to the pathogenesis of Alzheimer‘s Disease. Aquaporin 4 (AQP4) is a water channel protein expressed in the ependymal lining of the ventricular surface. We hypothesized that time‐dependent AQP4 expression would be altered in the lateral ventricle and cortex of a transgenic mouse model which expresses human amyloid precursor protein, corresponding with Alzheimer‘s Disease progression. Human Aβ expression was examined by immunohistochemistry in four brain quadrants in Tg2576 mice at 3, 8, 12, and 16 months. The average number of Aβ plaques, area per plaque, and percent of brain area covered by plaques significantly increased by 16 months. Lateral ventricles were enlarged in 12 and 16 month Tg2576 mice, concomitant with basal cell hyperplasia around the ventricles. AQP4 expression was observed on the apical and basolateral surfaces of basal cells surrounding the ventricles in 12 and 16 month mice, whereas AQP4 subcellular staining was restricted to the basolateral surface of basal cells at earlier time points and in controls. The altered pattern and distribution of AQP4 expression coincident with an increased number and area of plaques may be indicative of a change in fluid dynamics in later Alzheimer‘s Disease. This research was funded by an APS Summer Undergraduate Research Fellowship and by the University of Dayton Honors and Scholars Program.
The hepatic aquaporin 9 (AQP9) expression is regulated in response to castration and PPARα‐ agonist treatment
Authors: Lebeck Janne , Skowronski Mariusz Tomasz , Praetorius Jeppe , Frøkiær Jørgen , Nielsen Søren
Published: June 2021

It has previously been shown that male rats have higher hepatic expression of AQP9 than female rats (Nicchia et al, J Histochem Cytochem, 2001). The regulation of hepatic AQP9 has also been related to PPARα (Patsouris et al, J Clin Invest, 2004). This study aims to investigate the role of castration and PPARα–agonist (Wy 14643) administration in regulation of hepatic AQP9 protein expression in male rats. In castrated rats, immunoblotting showed a 1.7 fold increase in hepatic AQP9 (p < 0.01). This finding was confirmed by immunohistochemical analysis. Serum testosterone was decreased in the castrated group (p < 0.01), while serum insulin was 1.0 ± 0.3 and 1.9 ± 0.6 in castrated and controls, respectively, however this was not statistically significant and plasma glucose levels remained unchanged. In rats treated for 10 days with Wy 14643 (3 mg/kg/day), immunoblotting showed a 2.6‐fold decrease in the expression of hepatic AQP9 (p < 0.001). Immunohistochemical analysis revealed a marked decrease of AQP9 in the periportal zone, whereas AQP9 expressed in the perivenous zone appeared unchanged. There were no changes in serum insulin or glucose levels. We conclude that hepatic AQP9 protein expression increases in response to castration in male rats, whereas treatment with PPARα–agonist results in a marked decrease and redistribution of hepatic AQP9 protein.

Supported by the Danish National Research Foundation
Deletion of PKCalpha in mice attenuates the thrombin‐induced increase in lung vascular permeability
Authors: Vandenbroucke Emily Elizabeth , Vogel Stephen , Gao Xiaopei , Komarova Yulia , Malik Asrar B
Published: June 2021

We addressed the role of protein kinase Cα (PKCα) in signaling the increase in lung vascular permeability induced by the mediator thrombin. We used PKCα knockout mice to determine the effect of PKCα deletion on changes in endothelial permeability and adherens junction proteins. We determined microvessel filtration coefficient (Kfc) using the isolated‐perfused mouse lung preparation. Stimulation with PAR‐1 agonist peptide (10 μM) increased Kfc 50% less in the knockout mice than control wild type mice. We quantified vascular endothelial (VE) cadherin levels in mouse lung endothelial cells (MLECs) by Western blot analysis. Thrombin rapidly diminished VE‐cadherin levels in MLECs. PKCα deletion abrogated the VE‐cadherin disruption induced by thrombin. As PKCα activation was known to inhibit Akt, we observed higher Akt activity in PKCα‐deleted MLECs. We also observed that activated PKCζ was markedly increased in PKCα‐deleted MLECs, suggesting compensatory activation of PKCζ. The data demonstrate that PKCα is required to elicit the increase in lung vascular permeability. The mechanism of PKCα‐mediated increase in vascular permeability may involve signaling at the level of VE‐cadherin that promotes the disassembly of adherens junctions.

This study is supported by NIH # HL45638
Cholesterol regulates β‐adrenergic enhancement of L‐type Ca current in rat ventricular myocytes
Authors: Ochi Rikuo , Tsujikawa Hiroto , Okada Takao , Wolin Michael S , Gupte Sachin A
Published: June 2021

Cholesterol is a primary constituent of the plasma membrane, including lipid rafts/caveolae where various G protein‐coupled receptors colocalize with signaling proteins and channels. We studied roles of cholesterol in the β‐adrenergic modulatinon of L‐type Ca2+ currents (ICa,L) in rat ventricular myocytes using methyl‐β‐cyclodextrin (CD) and its cholesterol‐saturated complex (CD:chol). Exposing the myocytes to 10 mM‐CD for 10 min decreased cellular cholesterol content by 50%, while that to 5 mM CD:chol increased it by 40%. Isoproterenol (ISO) increased ICa,L 2.6‐fold and caused a negative shift in V0.5. Extracellular application of 10 mM‐CD for 10 min depressed the ISO‐induced ICa,L increase to 1.9‐fold and dialysis of 30 mM‐CD from pipette solution for 10 min suppressed the increase to 1.2‐fold. This dialysis‐induced suppression was recovered to 1.8‐fold by simultaneous superfusion of 15 mM CD:chol. However, dialysis of 30 mM CD:chol alone depressed significantly the ISO‐induced ICa,L increase to 1.7‐fold. These result suggest that normal density of cholesterol in the membrane is optimal for the β‐adrenergic increase in ICa,L. Cholsterol may regulate structural coupling of L‐type Ca2+ channels and adjacent regulatory proteins.
Angiotensin‐(1–7) opposes agonist‐induced constriction in endothelium denuded rat aortic rings via NO and PI3‐Kinase pathways
Authors: Allahdadi Kyan James , Leite Romulo , Webb R. Clinton , Santos Robson A. S.
Published: June 2021

Background: Angiotensin‐(1–7) [Ang‐(1–7)] is a potent vasodilator eliciting its effects primarily through endothelial derived factors. However, a direct effect of Ang‐(1–7) on the vascular smooth muscle (VSM) has not been fully investigated. This study tested the ability of Ang‐(1–7) to regulate agonist‐mediated VSM constriction in the absence of the endothelium.

Methods and Results: Endothelium denuded thoracic aortic rings (3 mm) from male Wistar rats (250–300g) were used in this study. Pretreatment with Ang‐(1–7) (10−5 M) significantly attenuated both Ang II and phenylephrine (PE) concentration response curves (10−10–10−6 M). This attenuated constrictor response was abolished by incubation with the selective Ang‐(1–7) receptor antagonist, A779 (10−5 M), the non‐selective nitric oxide synthase (NOS) inhibitor, L‐NAME (10−4 M), or the phosphatidylenositol 3‐kinase (PI3Kinase) inhibitor, LY29004 (10−5 M).

Conclusion: Our data suggest that Ang‐(1–7) activates its receptor and signals through NO derived from NOS isoforms within VSM. Additionally, we provided evidence that this opposed vasoconstrictor response involves the PI3‐kinase pathway. We suggest that this endothelium independent Ang‐(1–7)‐induced attenuation of the agonist‐mediated constriction of the VSM is an important mechanism by which Ang‐(1–7) participates in vascular reactivity and consequently blood pressure regulation.
Nox1‐mediated EGFR transactivation increases N‐cadherin shedding and smooth muscle migration
Authors: Miller Francis , Dammanahalli Jagadeesha , Banfi Botond , Takapoo Maysam , Bhalla Ramesh
Published: June 2021

The transmembrane glycoprotein N‐cadherin (N‐cad) has a critical role in the dynamic rearrangement of cell to cell adhesion. Reduction in the level of cell surface N‐cad by proteolytic shedding increases transcriptional activity of genes involved in cell migration. Although NADPH oxidases participate in cell growth, their role in shedding of N‐cad is unknown. We hypothesized that the NADPH oxidase subunit Nox1 contributes to shedding of N‐cad and migration of smooth muscle cells (SMC). Studies were performed in SMC cultured from wildtype (WT) and Nox1 null mice. Treatment of WT SMC with thrombin reduced membrane levels of N‐cad, which was prevented by the protease inhibitor MG132, suggesting N‐cad shedding. Pretreatment with antisense to Nox1 abolished the thrombin‐mediated shedding of N‐cad. Similarly, thrombin failed to reduce N‐cad levels in Nox1 null SMC. In WT SMC, cleavage of N‐cad and activation of matrix‐metalloproteinase (MMP)‐9 by thrombin was dependent on transactivation of EGFR. Activation of EGFR and MMP‐9 were significantly reduced in Nox1 null SMC. Finally, WT SMC migration was attenuated by the EGFR inhibitor AG1478 or by Nox1 antisense. These data suggest that Nox1‐mediated EGFR transactivation promotes proteolysis of N‐cad, potentially by MMP‐9, disrupting cell‐cell adhesions important in SMC migration.
PKG and SRF, Important Regulatory Factors Involved with Vascular Smooth Muscle Gene Expression
Authors: Brown Felricia Moyer , Choi Chung‐Sik , Lincoln Thomas M.
Published: June 2021

Vascular smooth muscle cell (VSMC) gene expression is altered in vascular diseases such as atherosclerosis. The NO/cGMP pathway appears to prevent the fibro‐proliferative response of VSMC in such instances. Cyclic GMP‐dependent protein kinase (PKG), the down‐stream mediator of NO/cGMP signaling, has been shown to regulate VSMC phenotype and stimulate VSMC gene expression. Serum response factor (SRF), a transcription factor associated with many signaling pathways including smooth muscle‐specific gene expression stimulates VSMC gene expression. The key objective of this study was to determine if PKG regulates SRF expression or translocation into the nucleus in VSMC. Results obtained: Primary cultures of VSMC were used to test whether PKG‐I activity increases SRF expression or translocation in VSMC. Down regulation of endogenous PKG‐I using siRNA decreased PKG‐I expression, but did not affect the overall level of SRF expression in VSMC. Over‐expression of PKG‐I caused increased SRF to be translocated to the nucleus, coincident with increased expression of smooth muscle myosin heavy chain. These effects were reversed in VSMC over‐expressing PKG‐I along with siRNA treatment. Conclusion: Up‐regulation of PKG‐I expression in VSMC may increase VSMC specific gene expression by increasing the SRF level in the nuclear compartment.

Supported by NIH grants: 5T32HL076125 and RO1HL53426
Serotonin Uptake in Veins, as Opposed to Arteries, Is Independent of the Serotonin Transporter
Authors: Linder A. Elizabeth , Ni Wei , Szasz Theodora , Burnett Robert , Watts Stephanie W.
Published: June 2021

Veins and arteries uptake serotonin (5‐hydroxytryptamine; 5‐HT). 5‐HT uptake in arteries depends on the 5‐HT transporter (5‐HTT), as it is inhibited by fluoxetine and fluvoxamine. The presence of 5‐HTT by immunohistochemistry was observed in vena cava. We hypothesized that 5‐HTT is responsible for 5‐HT uptake in veins. 5‐HT and its metabolite 5‐hydroxyindole acetic acid were measured by high performance liquid chromatography in vena cava (VC), jugular vein (JV), carotid artery (CA) and in aorta (RA) of rats treated with pargyline, an inhibitor of the enzyme responsible for 5‐HT degradation. After being exposed to exogenous 5‐HT (1 μ mol/L) for 15 min, an increase in 5‐HT content (ng/mg protein) was observed in VC (from 6.4 ± 0.9 to 16.5 ± 1.1), JV (4.0 ± 0.6 to 9.5 ± 0.9), RA (1.1 ± 0.2 to 6.4 ± 0.6.) and CA (0.9 ± 0.2 to 7.3 ± 0.7). Fluoxetine and/or fluvoxamine significantly inhibited 5‐HT uptake in RA and CA, but not in VC and JV. Western blot analysis showed lesser expression of 5‐HTT in VC than in RA. Moreover, studies performed in VC from mutant rats lacking the 5‐HTT showed no differences in 5‐HT uptake compared to VC from the wild type counterparts. These data suggest the 5‐HTT is not functional under physiological conditions in veins. The differences in 5‐HT handling between veins and arteries may represent alternative avenues for targeting the 5‐HT system in the peripheral circulation for controlling vascular tone.
PPARgamma Antagonists Attenuate Hypoxia‐induced Pulmonary Hypertension
Authors: Crossno Joseph T , Sullivan Timothy M , Morris Kenneth G , Klemm Dwight J
Published: June 2021

Rationale: Peroxisome proliferators‐activated receptor γ (PPARγ) agonists attenuate hypoxia (Hx)‐induced pulmonary artery (PA) remodeling. The present study tested the hypothesis that PPARγ antagonists exacerbate Hx‐induced PA remodeling.

Methods: Wistar‐Kyoto rats were randomized to normoxia (Nx) and hypobaric Hx and treated with or without the PPARγ agonist Rosiglitazone (ROSI) or the PPARγ antagonists GW9662, T0070907, or SR‐202 for 3 weeks.

Results: PA catheterization performed on Hx‐ROSI treated groups had no attenuation of Hx‐pulmonary hypertension (PH) vs Hx‐Controls. However, PPARγ antagonist treatment resulted in a decrease in PA pressures in response to chronic Hx [p < 0.05]. PA morphometric analysis demonstrated a marked decrease in proximal and distal PA remodeling vs Hx‐Controls [p = 0.003]. Right ventricular hypertrophy [RV/(LV+S)] was decreased in both the Hx‐PPARγ agonist and antagonist treated groups [p = 0.05]. The decrease in PA remodeling and RV hypertrophy was due to inhibition of proliferation without an increase in apoptosis.

Conclusions: PPARγ antagonists GW9662, T0070907 and SR‐202 exert a significant vascular protective effect on Hx‐induced PH and PA remodeling.
Overexpression of Angiotensin (1–7) – fusion protein attenuates monocrotaline (MCT) ‐ induced pulmonary hypertension
Authors: Shenoy Vinayak , Ferreira Anderson , Qi Yanfei , Dooies Kimberly Autumn , Raizada Mohan , Katovich Michael
Published: June 2021

Pulmonary hypertension is a progressive disease characterized by increased resistance to pulmonary blood flow. Our lab has previously demonstrated that ACE2 overexpression in the lungs of mice attenuated pulmonary pressure in monocrotaline (MCT) model of pulmonary hypertension. We therefore, wanted to investigate the effects of overexpression of Angiotensin (1–7) (Ang (1–7)), a product of ACE2 enzyme, in MCT‐induced rat model of pulmonary hypertension. Lentivirus producing Ang (1–7) ‐ fusion protein was injected into the right ventricle of male SD rats (250–300g). After two weeks of gene transfer, pulmonary hypertension was induced with subcutaneous injection of MCT (45mg/kg). After 4 weeks of MCT administration, rats were instrumented to measure right ventricular systolic pressure (RVSP) followed by heart excision to measure right ventricular hypertrophy. No significant difference in systemic blood pressure (tail cuff) was observed between the different treatment groups (control, 118 ± 6, MCT, 118 ± 3, MCT+Ang (1–7), 123 ± 4). However, MCT administration caused a significant increase in RVSP (MCT, 40 ± 4 mmHg, control 25 ± 2 mmHg, P<0.05) which was attenuated with Ang (1–7) treatment (28 ± 0.2 P<0.05 Vs MCT). MCT administration at the above dose and time period failed to induce right ventricular hypertrophy. We conclude that Ang (1–7) may hold therapeutic potential in the treatment of pulmonary hypertension.
EFFECTS OF CHRONIC VITAMIN C TREATMENT ON CARDIAC AND RENAL BAROREFLEXES IN RENOVASCULAR HYPERTENSION
Authors: Nishi Erika Emy , Oliveira‐Sales Elizabeth Barbosa , Bergamaschi Cassia Toledo , Campos Ruy Ribeiro
Published: June 2021

Oxidative stress seems to be involved in the development and maintenance of renovascular hypertension. The aim of the present study was to evaluate the effects of chronic administration of an antioxidant ‐ vitamin C (VitC) ‐ on arterial blood pressure (AP), baroreflex control of heart rate (HR) and on renal sympathetic nerve activity (RSNA) in the two kidney one clip hypertension (2K‐1C) model. Control and 2K‐1C male Wistar rats were treated with two different doses of VitC (150 and 250mg/kg/day) by gavage for seven days. VitC 150mg/kg decreased basal AP in 2K‐1C (23±7mmHg, n=13) as well as in control (C: 12±3mmHg, n=12) rats, with no changes in HR. In contrast, VitC 250mg/kg did not reduce significantly AP; however, there was a tendency of reduction in both groups. The baroreflex control of HR and RSNA was evaluated by bolus injection of phenylephrine. Both doses of VitC increased baroreflex sensibilitiy for bradicardic responses (C+Vit C150: 58%, n=6; 2K‐1C+VitC150: 47%, n=9; C+VitC250: 49%, n=8; 2K‐1C+VitC250: 128%, n=7) and RSNA (Cl+VitC150: 62%, n=6 and 2K‐1C+VitC150: 153%, n=6). Taken altogether, the present study suggests a possible role of oxidative stress in the baroreflex dysfunction in renovascular hypertension. Apparently, the actions of VitC on baroreflex control and on AP responses are independent. Supported by FAPESP.
Histopathophysiological Comparison Of Male And Female Heart Tissue Following Sustained Release Of Supraphysiological Levels Of Corticosteroid
Authors: Benghuzzi Ham A , Tucci Michelle A
Published: June 2021

Chronic stress results in elevated cortisol levels. Stress‐related increases in cortisol may be associated with known physiological abnormalities such as hypertension and CVD. The onset of CVD events differs between males and females, and it generally reported that stress and hormone status are responsible for these differences. The aim of this study was to investigate the heart tissue of adult male and female animals following twenty‐eight days of supraphysiological corticosterone levels. Animals were divided into four equal groups. Male group I and female group III animals served as control. Male in group II and female in group IV were surgically implanted with a TCP delivery device containing 50 mg corticosteroid. Twenty‐eight days post surgery, the heart were collected and processed for histological evaluations. No differences in heart wet weights were recorded between the groups. The length of the apex was increased following exposure to corticosterone in both male and female animals. Increases in the length of the ventricular bundles on the left side of the heart in the male animals exposed to corticosterone were observed. No differences were seen in the female animals in the ventricular bundle lengths when compared with female control animals. The data overall suggest the possibility of structural differences in the heart that are gender related and suggest hyperplasia.
Neural control of blood pressure and cardiovascular reactivity to stress in a spontaneously hypertensive mouse
Authors: Head Geoffrey A , Nguyen‐Huu Thu‐Phuc , La Greca Luisa , Shweta Amany
Published: June 2021

We investigated the contribution of the sympathetic nervous system (SNS) to spontaneous hypertension in the BPH/2J Schlager mouse. Male hypertensive (n = 6) and normotensive (BPN/3J; n = 8) mice were implanted with telemetry probes and recordings of cardiovascular and activity parameters made before and during aversive stress (shaker, restraint, cage‐swap) and non‐aversive stress (feeding). MAP was higher in BPH/2J than BPN/3J mice (128±1 vs 111±1 mmHg; P<0.001) as was the day‐night difference in MAP in BPH/2J animals (17±2 vs 6±1 mm Hg; P<0.001). Heart rate and activity were also greater in BPH/2J compared to BPN/3J (+19% and +88%, P<0.001). BPH/2J showed greater pressor responses to aversive stress (+70–100%) but a slightly greater pressor responses to feeding (+21%) compared with BPN/3J. Sympathetic blockade by pentolinium reduced blood pressure to comparable levels in both strains. The greater pressor response to aversive stimuli in BPH/2J mice but not feeding also suggests that the greater stress response observed in these animals is not due to a vascular amplifying effect which would likely affect all behavioural responses. Our findings of an exaggerated day‐night rhythms, responses to pentolinium as well as the hyper‐responsiveness to aversive stimuli suggests a major contribution from the SNS in the hypertension observed in this animal model.
Venous compliance in hypertensive adults
Authors: Delaney Erin P , Stillabower Michael E , Farquhar William B
Published: June 2021

Venous hemodynamic function is altered in animal models of hypertension. Lowered peripheral venous compliance (VC) may lead to a “centralization” of blood volume and contribute to the development of hypertension.

Purpose:

To investigate the effect of hypertension on VC in young and older adults. We hypothesized that limb VC would be lower in hypertensive (HTN) than normotensive (NTN) adults.

Methods:

VC was measure in 15 young NTN (YN; 23±1 years), 10 young HTN (YH; 23±1 years), 9 older NTN (ON; 65±1 years), and 11 older HTN (OH; 64±1 years) adults via venous occlusion plethysmography in the calf and forearm at baseline, during a cold pressor test, and after administration of nitroglycerin. Pressure‐volume relationships from a cuff deflation protocol were determined with quadratic regression; regression parameters were used to calculate VC at a pressure of 20 mmHg. Data were analyzed with an ANOVA.

Results

(mean±SE): There were no baseline differences in VC between YN and YH (Arm: YN=0.064±0.004 vs. YH=0.062±0.005 ml/dl/mmHg; Leg: YN=0.070±0.006 vs. YH=0.064±0.005 ml/dl/mmHg) or ON and OH (Arm: ON=0.046±0.004 vs. OH=0.049±0.004 ml/dl/mmHg; Leg: ON=0.050±0.005 vs. OH=0.050±0.006 ml/dl/mmHg). VC did not change from baseline in response to stressors (data not shown).

Conclusion:

These data indicate that otherwise healthy HTN adults do not demonstrate a reduction in peripheral VC below aged‐matched NTN adults.
VITAMIN C ADMINISTRATION REVERSES BLOOD PRESSURE INCREASE INDUCED BY SUCROSE FEEDING
Authors: Oliveira Thais Galvão Cesar , de Aquino Freitas Raphael Ribeiro , Oliveira‐Sales Elizabeth Barbosa , Boim Mirian Aparecida , Bergamaschi Cássia Toledo , Dolnikoff Miriam Sterman , Campos Ruy Ribeiro
Published: June 2021

Sucrose feeding (SF) induces oxidative stress, metabolic abnormalities and high blood pressure (HBP). The aim of this study was to investigate if dietary supplementation with vitamin C ameliorates HBP, hyperinsulinaemia and the mRNA renal expression levels of AT1, NAD(P)H oxidase subunits (p47phox and gp91phox), CuZnSOD induced by SF. Wistar rats (150–180g) were treated, for 30 days, with 20% sucrose solution (SC, n=15) instead of drinking water (CC, n=24). Ascorbic acid (AA, 150 mg/kg) was given by gavage to sucrose‐fed (SAA; n=17) or control rats (CAA; n=25) for the last 7 days of SF. Mean arterial pressure (MAP) was recorded on a Power Lab System. The mRNA expression levels were analyzed in the cortical renal tissue by Real Time RT‐ PCR. An increase (P<0.05) in MAP was observed in SC compared to CC (136±3; 111±4 mmHg). AA supplementation decreases (P<0.05) MAP either in CAA or SAA (102±3; 105±1 mmHg) rats. Basal glucose levels were unchanged but plasma insulin increased (P<0.05) after SF; no alterations were observed after AA. SF increased (P<0.05) p47phox and gp91phox expression but did not change CuZnSOD or AT1 mRNA expression. However, AA reversed the increase of mRNA expression of NAD(P)H oxidase subunits and it reduced (P<0.05) AT1 expression. CuZnSOD expression was unchanged after AA administration. These findings suggest AA ameliorates HBP probably by decreasing oxidative stress induced by SF. Supported by FAPESP (06/58219‐4).
Age and thermoregulatory blood flow during treadmill exercise
Authors: Ware Stuart K. , Ware Jason L.
Published: June 2021

The purpose of this study was to determine if age impacts the cutaneous vascular (thermoregulatory) response to exercise in women. Seven healthy women ages 20–24 were studied and compared to a group of seven healthy women ages 51–58. The exercise consisted of 20 min of brisk treadmill walking at a room temperature of 32 deg Celsius and a relative humidity of 45%. Skin blood flow (SBF) at the anterior forearm (dominant arm) and mid‐back was recorded immediately before and then throughout the exercise. A dual‐channel laser Doppler flowmeter was used to measure the SBFs, and the values were stored in computer and later analyzed by commercial software. Using ANOVA procedures mean SBFs for both skin locations were then compared before and at 2 min intervals during the exercise. The responses in the two age groups were then compared to determine if age had an effect on the vascular responses to exercise. Results showed vasodilation at each skin location in both groups with exercise. When the two groups were compared, it was found that the vascular responses were most pronounced in the younger group. The forearm SBF was 23% higher in the young group (P<0.05), and it was 34% higher at the back (P<0.05). The differential vascular responses to exercise between the two groups are likely explained by the effects of age on the structure and function of the cutaneous microcirculatory bed.
Hand‐Wrist Vascular Compliance as Assessed by Volume Changes with Gravity
Authors: Kim Jong Moon , Kraus Emily Ann , Gabel Brandon Clark‐Meyer , Hargens Alan Robert
Published: June 2021

Vascular compliance is a measure of an elastic deformation and is measured by altered volume and pressure. Our goal was to provide a simple method to examine age and gender effects on vascular compliance. Hand‐wrist volume was measured with 3% alcohol in distilled water (more accurate due to the decrease in surface tension) during two randomized conditions: 1) arm lowered for 5 min to allow for normal local pressure and volume, and 2) arm raised for 5 min to reduce local pressure and to induce a blood volume shift out of the elevated limb. Blood flow was occluded by an arm cuff to prevent arterial and venous flow. Percent differences in volume between the two experimental conditions were compared and significance was set at p<0.05. All subjects showed higher volume of displaced blood in the arm‐lowered position (463.34±13.60 ml) than that in the arm‐raised position (447.74±13.48 ml) (p<0.001). Percent difference in hand‐wrist volume in the young group (8 males and 8 females) was significantly greater (4.11±0.61%) than in the old group (2.85±0.61%) (9 males and 10 females) (p=0.017), but no statistically significant difference was found in gender comparison (p=0.062). Thus, hand volumetry is a quick, simple and potentially‐valuable method to measure blood volume shifts in the hand and wrist with gravity, and it can provide clinical information about cardiovascular risk in aged populations. Supported by NASA grant NNJ04HF71G.
Upregulation of TRPV4 channels in pulmonary arteries (PAs) contribute to chronic hypoxia induced myogenic tone and pulmonary hypertension
Authors: Yang Xiao‐Ru , Hughes Jennifer M. , Cao Yuan‐Ning , Flavahan Nicholas A. , Liedtke Wolfgang , Sham James S.K.
Published: June 2021

Acute reduction in alveolar O2 causes increase in pulmonary arterial pressure (Ppa) and prolonged hypoxia leads to vascular remodeling and pulmonary hypertension. The initial increase in Ppa may act as a stimulus for the development of pulmonary hypertension, possibly involving mechanosensitive cation channels. We have previously shown that the mechanosensitive TRPV4 channels are highly expressed in rat pulmonary arterial smooth muscle cells (PASMCs). Here we found that the TRPV4 agonist 4α‐PDD and hypotonicity induced significant increase in [Ca2+]i in PASMCs. These responses were inhibited by TRPV4 specific siRNA and the TRPV blocker ruthenium red. Real‐time RT‐PCR and western blot showed that TRPV4 expression was upregulated in PAs. Moreover, 4α‐PDD and hypotonicity induced Ca2+ responses were enhanced in PASMCs of rats after 4 weeks exposure to 10% O2. Significant myogenic tone, sensitive to ruthenium red, was also observed in pressurized pulmonary microvessels (100–200 ID, ≥35 mmHg) of chronic hypoxic but not normoxic rats. In extension of these results, the increase in Ppa (indexed by RVSP) and right heart hypertrophy were significantly attenuated in trpv4‐/‐ mice exposed to 1, 2, and 4 weeks of hypoxia. These results suggest the novel concept that TRPV4 channels are upregulated in PASMCs during chronic hypoxia and play significant roles in the development of myogenic tone and pulmonary hypertension.