bluuebunny/crossref_metadata_2025 huggingface dataset:
Age but few other commonly measured physiologic traits are associated with cardiovascular reactivity across multiple stressors in healthy young adults
Authors: Allen Alexander R , Gullixson Leah R , Liu Zhong , Eisenach John H
Published: June 2021

Normotensive individuals who show a robust pressor response to sympathoexcitatory stimuli are at increased risk for developing hypertension. In healthy young adults, we defined a cumulative pressor score (CPS) as the mean increase in systolic arterial pressure across 3 stressors: mental stress, cold pressor test (CPT), and isometric handgrip to fatigue (HG). We hypothesized that individuals with high CPS would possess other physiologic traits associated with cardiovascular reactivity. In 143 subjects (M/F: 57/86, ages 18–40 yr) quartiles were formed with hyper‐responders (CPS ≥ 27.0 mmHg) in the top and hypo‐responders (≤ 16.6 mmHg) in the bottom quartile. Comparisons across quartiles included age, sex, BMI, arterial catecholamines at rest and during stress, HR variability, baroreflex sensitivity by modified Oxford, pulse wave velocity, and 24‐hr ambulatory BP measures. Age was associated with CPS, as hyper‐responders (mean age±SE: 29±1 yr) were approximately 4 yrs older than each quartile (p<0.01). Hypo‐responders had decreased norepinephrine during CPT (p<0.01) and HG (p<0.01), and epinephrine during HG (p<0.01) when compared to each quartile. No other physiologic variable was associated with CPS or quartile. These findings imply that while hyper‐responders may be at greatest cardiovascular risk, it appears to be a consequence of normal aging, as few other intermediate physiologic markers of BP reactivity were associated with pressor scores. Support: HL‐089331, CTSA RR‐024150
Cardiovagal baroreceptor sensitivity and peak oxygen consumption in chronic obstructive pulmonary disease (COPD)
Authors: Edgell Heather , Steinback Craig , Reiger Mathew , Vogan Norah , Wong Eric , Bhutani Mohit , Stickland Michael
Published: June 2021

Patients with chronic obstructive pulmonary disease (COPD) typically have reduced exercise capacity (VO2peak) and cardiovagal baroreflex sensitivity (BRS). The underlying mechanism for lower BRS in COPD is unclear. Exercise training has been shown to increase VO2peak and BRS sensitivity. Therefore, it was hypothesized that lower BRS in COPD would be related to VO2peak. COPD patients (n=14; Mean FEV1=59.4±33.9%predicted, age=70.9±8.8) and healthy controls (n=8; Mean FEV1=104.0±12.5%predicted, age=67.5±5.1) were recruited. None had diagnosed cardiovascular disease, diabetes or sleep apnea. VO2peak data were obtained from a cardiopulmonary exercise stress test. On a separate day resting heart rate and beat‐by‐beat blood pressure were measured. BRS was determined using the sequence method. We found lower BRS (7.2±3.8 vs. 14.8±9.7ms/mmHg; P=0.01) and VO2peak (19.6±4.0 vs. 31.1±9.3mL/kg/min; P<0.001) in COPD, and a linear relationship in COPD and controls between VO2peak and BRS was observed (R2=0.24; P=0.02). Preliminary results indicate that the reduction of BRS is related to reduced exercise capacity in COPD. Funded by Canadian Institutes of Health Research.
Metabolic and cardiovascular effects of AT1 receptor blockade in rats with diet‐induced obesity
Authors: Junior Silvio Assis Oliveira , Guizoni Daniele Mendes , Martinez Paula Felippe , Torres Bárbara Pereira , Campana Alvaro Oscar , Padovani Carlos Roberto , Okoshi Katashi , Okoshi Marina Politi , Cicogna Antonio Carlos
Published: June 2021

Causes of metabolic and cardiovascular disturbances in obesity are poorly understood. This study analyzed whether the angiotensin II receptor blockade reverses abnormalities in diet‐induced obesity. Wistar‐Kyoto rats (n=40) were subjected to control (C) or hypercaloric diet (OB) for 30 weeks and then assigned to four groups: C, C+Los, OB, and OB+Los. Los‐groups received losartan (30mg/kg/day) during five weeks. Subsequently, cardiac structures and function were assessed by echocardiography. Glycemia was studied by tolerance glycemic curve. OB‐groups presented higher body mass and adiposity. Other results are in the table.

Conclusion

Losartan improves metabolic and cardiovascular profile in diet‐induced obesity.
Thrombospondin‐2 may exert its effects on fetoplacental development in the BPH/5 mouse model of preeclampsia (PE) through CD36 and CD47 receptors
Authors: Zhou Yi , Sones Jenny L. , Woods Ashley K. , Davisson Robin L.
Published: June 2021

The BPH/5 mouse model spontaneously develops the hallmarks of PE, including fetoplacental abnormalities and an imbalance in pro‐and anti‐angiogenic factors. We have utilized ultrasonography to categorize BPH/5 fetoplacental units (FPUs) at e10.5 into three increasingly severe phenotypic classes (I, II and III). We previously demonstrated that thrombospondin 2 (Thbs2), an inhibitor of angiogenesis, is significantly increased in BPH/5 Class II and III FPUs compared to C57. To further understand the mechanism of Thbs2 in BPH/5 FPUs, we hypothesized that Thbs2 could either activate apoptosis signaling pathways via CD36 or block angiogenesis signaling through interaction of CD47 and VEGFR2. Real time qPCR results show that CD36 mRNA expression was significantly increased in BPH/5 Class II and III FPUs (II=2.8 ± 0.42; III=3.2 ± 0.1 fold vs C57, n=3, p<0.05), while CD47 (1.5 ± 0.15 fold vs C57, n=3, p<0.05) and VEGFR2 (1.7 ± 0.17 fold vs C57, n=3, p<0.05) were up‐regulated in BPH/5 Class II FPUs. Western analysis confirmed that CD47 protein expression was increased in BPH/5 FPUs. We conclude that Thbs2 may exert its effects on fetoplacental development in BPH/5 via CD36 and CD47 receptors. Funding: QNRF NPRP09‐1099‐279.
Strain‐dependent variation in vasoreactivity in isolated mouse thoracic aorta
Authors: Kim Seung Kyum , Avila Joshua J. , Massett Michael P.
Published: June 2021

Understanding genetic influence on vascular reactivity is important for identifying genes underlying impaired vascular function. Therefore, the aim of this study was to characterize genetic variation in vascular reactivity using 14 inbred strains of mice. Concentration response curves to phenylephrine (PE), potassium chloride (KCl), acetylcholine (ACh), and sodium nitroprusside (SNP) were obtained from aortic rings from male mice (n ≈ 4/strain). Contractile responses to PE and KCl varied significantly across strains. In response to PE, aorta from PWD/PhJ had the lowest (43.8 ± 6.7 %) and aorta from NZW/LacJ had the highest (160.8 ± 15.6 %) maximal response. Aorta from C57BL/6J and 129S1/SvImJ had the lowest (112.0 ± 19.8 %) and the highest (183.2 ± 5.5 %) maximal response to KCl, respectively. Endothelium‐dependent relaxation responses to ACh were also significantly different across strains with NZW/LacJ having the lowest (50.1 ± 7.7 %) and C3H/HeJ having the highest (98.6 ± 0.5 %) maximal response. Maximal endothelium‐independent relaxation responses to SNP were similar among strains. Differences in sensitivity to all agents were observed across strains. These data demonstrate that there are large strain‐dependent differences in vasoreactivity across inbred mouse strains and indicates that genetic background influences vascular function.

Supported by NIH grant HL085918 to MPM
Competitive Exchange Diffusion to determine transport of hOCT2 inhibitors
Authors: Harper Jaclyn Nicole , Wright Stephen H.
Published: June 2021

The organic cation (OC) transporter 2 (OCT2; SLC22A2) is the entry step of the OC secretory pathway of human renal proximal tubules. We used Competitive Exchange Diffusion (CED) to indirectly assess whether inhibitors of OCT2‐mediated transport also serve as substrates for transport. In CED, CHO cells that stably expressed hOCT2 were preloaded with [3H]MPP (‘M*’) to steady‐state (30 min). The solution was then replaced with one containing either (i) the pre‐loading solution (PLS), or (ii) PLS plus unlabeled test ligand. Whereas PLS alone supported no change in M* content over 5 min, presence of a transported ligand supported CED and a decrease in M* content. When the ligand was 100 uM unlabeled MPP (20× its Kt), M* decreased by 40% within 1 min. Saturating concentrations of the other OCT2 substrates (20× their Kt values), TEA, metformin and cimetidine supported M* efflux comparable to that supported by MPP (10 mM glucose, sucrose, or mannitol supported no CED). We then tested two compounds recently identified as strong inhibitors of OCT2 (Kido et al, J Med Chem, 54(13):4548–58, 2011): the sterol, adrenosterone (AS; IC50 of 6 uM) and the strong base, phenyltoloxamine (PT; IC50 of 6 uM). Interestingly, saturating AS or PT did not support CED‐induced efflux of M*. AS and PT both proved to be non‐competitive inhibitors of OCT2‐mediated MPP transport (decreasing Jmax with little or no change in Kt). NIH grant 5R01DK58251.
Impact of Antenatal Betamethasone (B) on p47 phox in Kidney and 8‐isoprostane Responses to Angiotensin II in Proximal Tubule Cells in Male Sheep after Uninephrectomy
Authors: Bi Jianli , Su Yixin , Figueroa Jorge , Chappell Mark C. , Rose James C.
Published: June 2021

Antenatal B administration reduces nephron number and causes hypertension in offspring. It is not clear if antenatal B enhances responses to a second insult (additional loss of nephrons).

This study examined if antenatal betamethasone exposure increases susceptibility to a second “hit”. Adult male sheep exposed to B or Vehicle (V) (n=3–6) before birth underwent uninephrectomy (UN). Necropsy was performed 15 days after UN. Renal cortex was collected at both times. PTCs from both kidneys were stimulated with Ang II with or without its antagonists. P47 expression was determined by western blotting and 8‐PGF in media was measured by ELISA. Data were analyzed by ANOVA or T‐test.

B exposure increased p47 expression (p<0.05) in kidneys obtained at uninephrectomy (first kidney) and at necropsy (second kidney) (B sheep vs. V sheep F=16, P=0.007). UN increased p47 in the second kidney in both groups (F=6.5, p=0.043). The highest expression of p47 was in the second kidney from the B exposed animals.

Ang II stimulated 8‐PGF secretion from PTCs of the kidneys from B exposed sheep only (F=8.4, P=0.009 first kidney) and (F=5.5, P=0.044 second kidney). The response was blocked by candesartan but not PD123319. Ang II increased 8‐PGF secretion the most in PTCs from second kidney of B exposed animals (p<0.05).

This suggests antenatal B exposure predisposes the kidney to oxidative stress from a“second hit”. Supported by HD‐47584 and HD‐17644
Oleanolic acid‐induced regulation of atrial natriuretic peptide secretion in perfused beating atrial model
Authors: Kim Hye Yoom , Li Xiang , Cho Kyung Woo , Kang Dae Gill , Lee Ho Sub
Published: June 2021

The heart is an endocrine organ involved in the regulation of blood pressure and body fluid homeostasis through the secretion of atrial natriuretic peptide (ANP). Oleanolic acid (OA) is a well known triterpenoid compound that exist widely in food and medicinal herbs. The purpose of present study was to examine the effect of OA in the regulation of ANP secretion. Experments were performed in beating rat atria. OA (30 mg/kg/day) was administrated orally in Sprague‐Dawley rats : Atrial dynamics, pulse pressure, and ANP secretion were measured. OA administration increased ANP levels in plasma and atria. To identify the effects of OA on the ANP secretion by activation of ¥â‐adrenergic and muscarinic receptor, isoproterenol and acetylcholine were administration in the atria from OA‐ and sham‐treatie rats. Oral intake of OA accenturated the acetylcholine‐induced increase of ANP secretion. However, OA attenuated the isoproterenol‐induced decrease of ANP secretion. These findings demonstrate that OA attenuates the decrease of ANP secretion induced by activation of ¥â‐adrenergic receptor and accentuates the increase of ANP secretion induced by muscarinic receptor. These results suggest that oral intake of OA induces an activation of the ANP system in rats.
Collecting Duct Renin Synthesis and Secretion are Stimulated by Angiotensin (Ang) II via Protein Kinase C (PKC) Activation and cAMP Accumulation
Authors: Liu Liu , Lara Lucienne S. , Gonzalez Alexis , Bourgeois Camille RT , Seth Dale M , Navar L. Gabriel , Prieto Minolfa C.
Published: June 2021

In rats and mice, Ang II type 1 receptor (AT1R) activation stimulates renin synthesis via PKC activation in the rat inner medulla CD (IMCD) cells. Because cAMP is an important second messenger for the Ang II signal transduction, we hypothesized that Ang II stimulates CD renin synthesis and release via PKC activation and cAMP accumulation. In mouse cortical CD cell line (M‐1 cells) treated with Ang II (8 hrs) we found increased renin mRNA in a dose‐dependent manner. PKC inhibition with Calphostin prevented this response. Likewise, Ang II increased renin and prorenin protein levels at 15 min with a sustained response for 2 hrs. Renin content measured in the cell culture media augmented in Ang II‐treated cells by 6 hrs (Ang II: 31±4 vs. 19±4 ng Ang I/hr/ml; p<0.05). Importantly, Ang II (10−7M) increased cAMP levels by 1 min (148±32 pmol/mg protein) reaching a peak at 30 min (290±27 vs. 44±0 pmol/mg protein; p<0.05). This response was prevented by PKA inhibition with H89 or PKI. Forskolin, an adenlyate cyclase activator, increased renin transcript in a dose‐dependent manner. Taken together, Ang II stimulates renin synthesis and secretion via PKC activation and cAMP stimulation in M‐1 cells; suggesting that during Ang II‐dependent hypertension, increased apical secretion of renin by the CD cells into the luminal fluid may augment intratubular Ang II formation, thus contributing to enhance distal nephron sodium reabsorption.
Angiotensin II increases Transient Receptor Potential Vanilloid 4 channel Expression and Phosphorylation in Hypothalamic Cell line 4B
Authors: Saxena Ashwini , Nedungadi Thekkethil Prashant , Carreno Flavia , Bachelor Martha , Cunningham J. Thomas
Published: June 2021

We have previously demonstrated that in bile duct ligated rats, an animal model of inappropriate vasopressin (AVP) release, TRPV4 protein expression and membrane trafficking is increased in AVP neurons. Here, we used an in vitro approach with an immortalized AVP expressing cell line (4B cells) to investigate the possible regulation of TRPV4 by angiotensin II (Ang II), utilizing real‐time polymerase chain reaction (RT‐PCR), Western Blot (WB) and immunohistochemistry (IHC). We have characterized, for the first time, the presence of TRPV4 mRNA and protein 4B cells. After Ang II (1μM;1 hr) treatment, TRPV4 mRNA increased nearly 5‐ fold (Control vs. Ang II; 1.12±0.28 vs. 4.98±0.65; p<0.001). We noted increased TRPV4 immunoreactivity after 1 and 2 hours of Ang II treatment. Western Blot analysis showed significantly (p<0.001) increased TRPV4 levels in crude membrane fractions of Ang II treated cells compared to vehicle controls. Also, WB analysis revealed increased tyrosine phosphorylation of TRPV4 following Ang II treatment as compared to vehicle treated cells (p<0.001). Thus, 4B cells may be an appropriate in vitro model to investigate the translational and posttranslational regulation of TRPV4 in hypothalamic neurons.
Simulated microgravity modifies intestinal epithelial barrier function and alters expression of tight junction proteins
Authors: Stork Cheryl Anne , Marchelletta Ronald R. , Prisk G. Kim , McCole Declan F.
Published: June 2021

The integrity of the intestinal epithelial barrier is tight junctiondependent. Previous studies showed that exposure to microgravity compromises the human immune system and increases enteropathogen virulence. However, the effects of microgravity on epithelial barrier function are unknown. Here, we investigated the effects of simulated microgravity on HT‐29 intestinal epithelial cell (IEC) barrier properties. IECs were (i) cultured on microcarrier beads for 18 days while subjected to simulated microgravity in a rotating wall vessel (RWV); (ii) cultured on beads for 18 days in culture plates (static control); (iii) grown in flasks for 18 days (flask control); prior to seeding on inserts. RWV cells displayed reduced transepithelial resistance (TER) on days 2–14 after seeding (−6 ± 3% vs. static day 11; n=4). Confocal analysis revealed increased expression of tight junction proteins Zonula Occludens‐1 (ZO‐1) and Occludin in RWV cells over 2–14 days (ZO‐1: 59–66 ± 11 pixels/unit area; n=3–4) vs. static (40–56 ± 10 p/ua) and flask (38–46 ± 10 p/ua) controls. Moreover, RWV cells showed delayed localization of Occludin and ZO‐1 to the apical junctions suggesting altered intracellular trafficking. In conclusion, cells exposed to simulated microgravity exhibit reduced TER and altered expression and distribution of tight junction proteins suggestive of a functional barrier defect.

Supported by NIH UH2 AA019708‐01.
Endothelins induce choleresis through ETB receptors coupled to nitric oxide release and vago‐vagal reflexes
Authors: RODRIGUEZ MYRIAN ROXANA , Ventimiglia María Silvia , Najenson Ana Clara , Vatta Marcelo Sergio , Bianciotti Liliana Graciela
Published: June 2021

We previously showed that brain applied ET‐1 and ET‐3 in rats induce choleresis or cholestasis depending on the dose. In this study we evaluated if endothelins (ETs) regulated bile secretion when peripherally infused. Animals were prepared with jugular vein and bile duct cannulation to infuse drugs and collect bile samples. At the doses used ETs did not affect hepatic blood flow or portal venous pressure. ETs induced significant choleresis, being ET‐1 more potent than ET‐3. ETs increased bile salts, electrolytes and glutathione excretion supporting enhanced bile acid dependent and independent flows. ETs‐induced choleresis was mediated by ETB receptors coupled to nitric oxide and abolished by truncal vagotomy or capsaicin perivagal application. RT‐PCR and western blot analysis showed ETA and ETB receptor expression in the vagus nerve. ETs through ETB stimulated sodium taurocholate co transporter polypeptide (Ntcp) and aquaporin 8 (AQP8) translocation to the plasma membrane and augmented Ntcp, AQP8, bile salt export pump (Bsep) and multidrug resistance protein 2 (Mrp2) in microsomal membranes. Real time PCR studies showed that exposure to ETs increased the mRNAs of these transporters. ETs induced choleresis through ETB receptors coupled to nitric oxide and vago‐vagal reflexes without involving hemodynamic changes. These findings suggest that ETs may play a beneficial role when bile secretion is impaired.

(CONICET PIP 0370/ ANPCyT‐PICT2008‐0716)
Increased serum bile acids after extrahepatic biliary obstruction causes leakiness to the blood brain barrier via the disruption of tight junctions
Authors: DeMorrow Sharon , Frampton Gabriel , Galindo Cheryl , Pae Hae Yong , Quinn Matthew
Published: June 2021

The brain is protected by the blood brain barrier (BBB), which becomes leaky during obstructive cholestasis via an unknown mechanism. A common feature of cholestasis is increased serum bile acids, which can signal via the membrane bound receptor, TGR5. Our aim was to determine the effects of bile acids in the permeability of the BBB during the course of cholestasis. After bile duct ligation (BDL) in the rat, serum bile acid concentrations increased which was paralleled by increased BBB permeability as shown by an Evan's blue exclusion assay. In vitro, there was increased permeability of a monolayer of rat brain microvessel endothelial cells (RBMEC) after treatment with serum from BDL animals compared to controls and also after treatment with cholic acid (CA), deoxycholate (DCA) and chenodeoxycholate (CDCA), but not after ursodeoxycholate (UDCA). In parallel, the TGR5 agonist betulinic acid also increased permeability in vitro. Analysis of RBMECs treated with CA, CDCA, DCA, and betulinic acid all exhibited a disorganized cytoskeleton (demonstrated by F‐Actin staining) and an altered distribution of the tight junction proteins ZO‐1 and ZO‐2. Our data suggests that the increased circulating serum bile acids contribute to the increased permeability of the BBB seen during obstructive cholestasis. This is possibly via a mechanism involving TGR5 activation and subsequent disruption of the tight junctions.
Myocardial cholesterol homeostasis is altered by age and Cav‐3 knockdown
Authors: Schilling Jan Mirko , Fridolfsson Heidi N , Panneerselvam Mathivadhani , Finley Joseph C , Kellerhals Sarah E , Niesman Ingrid , Head Brian P , Roth David M , Patel Hemal H
Published: June 2021

Cardiovascular disease (CVD) is a leading cause of mortality in the United States. The percentage of people over the age of 65 years is increasing dramatically and by 2050 the elderly will account for >2 billion people worldwide. 80% of deaths caused by CVD occur in patients 65 years of age and older. Caveolae are membrane microdomains enriched in cholesterol, glycosphingolipids, sphingomyelin, and the protein caveolin and are critical to ischemic tolerance. Both, caveolin‐3 (Cav‐3) and cholesterol are key to the formation and maintenance of caveolae. We hypothesized, that cellular cholesterol homeostasis is altered in aged wild type (WT) and Cav‐3 knock out (KO) mice. Our data show that 1) aged hearts have a reduced number of caveolae and tolerance to ischemia, 2) gene array data show altered expression on genes involved in cholesterol biosynthesis, transport/processing, and transcriptional regulation with age and Cav‐3 knockdown, and 3) cholesterol is reduced in membrane homogenates of aged hearts and Cav‐3 KO mice. Therefore, membrane cholesterol and Cav‐3 play key roles in the response to ischemic stress and altering these components may be lead to better ischemic tolerance in the aged heart.
Sex differences in cannabinoid antinociception against inflammatory pain
Authors: Craft Rebecca M , Kandasamy Ram
Published: June 2021

We examined sex differences in antinociceptive and anti‐inflammatory effects of delta‐9‐tetrahydrocannabinol (THC) using the complete Freund's adjuvant (CFA) model of inflammatory pain (hindpaw CFA injection). Gonadally intact adult rats received THC systemically (0, 0.32, 10, 3.2 mg/kg i.p.) or intraplantar (0, 250, 500 μg i.pl.), on days 1, 3 and 7 post‐CFA. THC attenuated mechanical allodynia and thermal hyperalgesia in both sexes via both routes of administration, but tended to be more effective in females than males when given i.pl. THC also was more effective in females in reversing CFA‐suppressed drinking one day after CFA injection. In contrast, i.pl. THC decreased paw inflammation more in males. These results agree with previous studies demonstrating greater cannabinoid antinociception in females than males using acute pain models, and suggest that sex differences in the antinociceptive effects of THC in the CFA model are not related to its anti‐inflammatory properties. Additionally, i.pl. THC did not affect locomotor activity (or response latency in the uninflamed paw), indicating that greater antinociceptive effects of THC in females are not secondary to THC's greater sedative effect in females (observed when THC is given systemically). These results suggest that cannabinoids may be more effective analgesics in females than males. Supported by the State of Washington Initiative Measure No. 171.
5‐hydroxy‐2‐(4‐hydroxy‐3‐methoxyphenyl)‐3,7‐dimethoxy‐4H‐chromen‐4‐one (MSF‐2) suppresses fMLP‐mediated respiratory burst in human neutrophils by inhib‐iting phosphatidylinositol 3‐kinase activity
Authors: Liao Chang-Hui , Chen Jih-Jung , Liu Chia-Hsin , Tseng Ching-Ping , Day Yuan-Ji
Published: June 2021

This study investigates the effect and the underlying mechanism of MSF‐2, a lignan extracted from the fruit of Melicope Semecarprifolia, on fMLP‐induced respiratory burst in human neutrophils and suggests a possible thera‐peutic approach to ameliorate disease associated with neutrophil hyperactivation. MSF‐2 inhibited fMLP‐induced neutrophil superoxide anion production, cathepsin G release and migration in human neutrophils isolated from healthy volunteers, reflect‐ing inhibition of phosphatidylinositol 3‐kinase (PI3K) activation. Specifically, PI3K/AKT activation results in migration, degranulation and superoxide anion pro‐duction in neutrophils. MSF‐2 suppresses PI3K activation and PIP3 pro‐duction, and consequently inhibits downstream activa‐tion of PDK1 and AKT. Further, PI3K also stimulates respiratory burst via PLC‐dependent elevation of intracellular calcium. MSF‐2 reduces fMLP‐mediated PLCγ2 activation and intracellular calcium accumulation notably through extracellular calcium influx in a PI3K and PLC‐dependent manner. However, MSF‐2 is not a com‐petitive or allosteric antagonist of fMLP. Additionally, in an in vivo study, MSF‐2 prevents fMLP‐induced neutrophil infiltration and inflammation in mice. In conclu‐sion, MSF‐2 opposes fMLP‐mediated neutrophil activation and inflammation by inhibiting PI3K activation and subsequent activation of AKT and PLCγ2.
The mechanism of the marine thromboxane B2 inhibitor Manzamine A: possible involvement of rat brain microglia p90 ribosomal S6 kinase 1
Authors: Clifford Jonathan A , Lach Joseph , Hall Mary L , Mayer Alejandro M
Published: June 2021

Manzamine A (MZA) potently inhibited LPS‐activated rat microglia (BMΦ) TXB2 (IC50<0.016 μM) and O2− (IC50=0.1 μM) generation, (BMC Pharmacology 5(1)6, 2005), as well as LPS‐activated human BMΦ TXB2 (apparent IC50<0.7 μM) (Inflam Res. 53(S3): S217, 2004). We have shown that MZA selectively inhibited rat p90 ribosomal S6 kinase 1 or RSK1 (IC50=15.01 μM) vs human RSK2 (IC50=108.4 μM) (Program 545.5, Society of Neuroscience, 2009). We hypothesized that TXB2 inhibition by MZA in BMΦ involved RSK1 inhibition. In order to test our hypothesis we first investigated the presence of RSK1 and RSK2 proteins in rat BMΦ. Briefly, we prepared protein extracts from untreated, LPS (0.3 ng/mL)‐treated, and LPS + PMA (1μM)‐ treated BMΦ, and probed by western blot analysis using RSK1 (Cell Signaling) and (phosphorylated RSK1) pRSK1 (Epitomics) antibodies.

Results

1) RSK1 and RSK2 proteins were present in similar quantity in all BMΦ extracts tested; 2) PMA enhanced pRSK1 protein in LPS‐treated BMΦ. Thus because RSK1, RSK2, and pRSK1 proteins are present in rat microglia they may be involved in TXB2 inhibition by MZA. We conclude that in order to test this hypothesis, the concentration‐dependent effect of MZA on BMΦ RSK1 phosphorylation and concomitant TXB2 inhibition will require further investigation. Support to JC and JL by the Student Fellowship Program, Office of Research, Midwestern University is gratefully acknowledged.
β3 integrin/PDGF receptor synergistic signaling mediates cardiac fibrosis in a mouse model of pressure overload hypertrophy
Authors: Balasubramanian Sundaravadivel , Kasiganesan Harinath , Quinones Lakeya , Bradshaw Amy , Kuppuswamy Dhandapani
Published: June 2021

The precise role of integrins in the process of cardiac fibrosis is not well understood. We hypothesized that upon cardiac hypertrophic stimulation, the synergistic activation of β3 integrin and platelet‐derived growth factor receptor (PDGFR) leading to nonreceptor tyrosine kinase (NTK) signaling in cardiac fibroblasts contributes to fibrosis. In response to pressure‐overload induced by transverse aortic constriction (TAC), β3 integrin knockout (β3 KO) mice exhibited reduced fibrillar collagen, fibronectin, and fibroblast specific protein (FSP1) levels in comparison to wild‐type (WT) mice. Isolated cardiac fibroblasts (Cfb) from β3 KO mice exhibited a reduction in cell adhesion and spreading as well as PDGF‐induced cell proliferation and migration when compared to Cfb from WT mice. Next, we analyzed the phosphorylation state of PDGFR and NTK upon PDGF stimulation in Cfb. Our data indicated a marked reduction in the activation of PDGFR, Pyk2, Src and FAK in β3 KO Cfb when compared to WT Cfb. These data suggest that β3 integrin provides a necessary co‐stimulation for fibrotic signaling in Cfb. Furthermore, our studies in the mouse TAC model where dasatinib, an inhibitor of PDGFR and NTK, was administered via a mini‐osmotic pump, exhibited a preserved ventricular geometry and function and reduced fibrosis upon 4 wk TAC. NTK inhibition with dasatinib also reduced the proliferation, migration and mitogenic signaling of Cfb. These data indicate that integrin‐PDGFR synergism could be a potential target to treat cardiac fibrosis.
ALTERNATIVELY SPLICED BNIP3 ISOFORM PROMOTES CELL SURVIVAL BY RECRUITING BCL‐2 TO THE IP3‐RECEPTOR
Authors: Gordon Joseph William , Hai Yan , Gang Hongying , Kirshenbaum Lorrie
Published: June 2021

Alternative splicing provides a versatile mechanism by which cells generate proteins with different or even antagonistic properties. We previously identified a novel splice variant of Bnip3 (Bnip3ΔEx3) generated in cardiac myocytes subjected to hypoxia. Like Full‐length Bnip3 (Bnip3FL), Bnip3ΔEx3 forms a complex with the antiapoptotic protein Bcl‐2; whereas, functional analysis of a Bnip3 mutant that lacks a Bcl‐2 interacting domain acts as a more potent inducer of programmed cell death, that can not be inhibited by Bnip3ΔEx3. In silico analysis of Bnip3ΔEx3 reveals an endoplasmic reticulum (ER) targeting domain within its C‐terminus, that is not present in Bnip3FL. We validated that Bnip3ΔEx3 is indeed localized to the ER and demonstrate that Bnip3ΔEx3 mutants that lack this ER targeting domain fail to antagonize cell death induced by Bnip3FL or hypoxia. Furthermore, we demonstrate that Bnip3ΔEx3 physically interacts with the IP3 receptor and serves to recruit Bcl‐2 to the ER; whereas, Bnip3ΔEx3 fails to protect against programmed cell death in the presence of a Bcl‐2 mutant that lacks its IP3 receptor interacting domain. Given the otherwise lethal consequences of de‐regulated Bnip3FL expression in post‐mitotic cells, our findings reveal a novel intrinsic defense mechanism that opposes the lethal consequences mitochondrial‐ER cross‐talk associated with programmed cell death in ventricular myocytes.
Leptin activates rat carotid body Type I cells and brainstem astroglial cells
Authors: Marina Nephtali , Kasymov Vitaliy , Mohamed-Ali Vidya , Kasparov Sergey , Gourine Alexander V
Published: June 2021

Obesity is strongly associated with significant increases in muscle vasoconstrictor sympathetic activity which may contribute to the development of obesity‐related hypertension. Sympathetic tone and blood pressure can be reflexly increased by activation of peripheral chemoreceptors in the Carotid Bodies. Similarly, we have recently shown that optogenetic activation of ventral medullary astrocytes results in sympathoexcitation and increases in blood pressure. Leptin and leptin receptors have been recently found to be expressed both in Carotid Body Type I cells and in astroglial cells. Here, we explored whether these receptors are functional and whether leptin is capable of triggering activation of Carotid Body Type I cells and brainstem astroglial cells in vitro.

RESULTS

In Carotid Body dissociated cultures and primary cultured brainstem astrocytes loaded with Ca2+ indicator Fura‐2, application of recombinant rat leptin evoked dose‐dependent increase in [Ca2+]i. The responses had long latency (approximately 500 sec.) and [Ca2+]i oscillations were sustained for prolonged periods.

CONCLUSIONS

These data suggest that sustained high levels of circulating leptin in obesity have the potential to activate Carotid Body Type I cells and brainstem astroglial cells, leading to increases in sympathetic tone and blood pressure.
Increasing leptin sensitivity increases blood pressure in mice on a TH2 responsive background only
Authors: de Chantemele Eric J Belin
Published: June 2021

Besides inducing sympatho‐activation, leptin promotes T cell‐mediated pro‐inflammatory cytokines release. Since T cells play a key role in hypertension we hypothesized that increasing leptin sensitivity via protein tyrosine phosphatase 1B (PTP1B) deletion or increasing leptin concentration would promote inflammation and hypertension through a T cell dependent mechanism. PTP1B was deleted in mice on either a TH1 (C57) or a TH2 (Balb/C) T cell background. Blood pressure (BP) recording showed that PTP1B deletion and leptin infusion elevated BP in TH2 responsive mice only. As reflected by the BP response to ganglionic blockade, sympathetic tone was increased in response to PTP1B deletion and leptin in TH2 mice. Plasma aldosterone levels, indexes of the activation of the renin‐angiotensin‐aldosterone system were also increased in response to leptin and PTP1B deletion in TH2 mice. PTP1B deletion suppressed TH2‐derived anti‐inflammatory cytokine secretion (IL‐10, IL‐4) and increased TH1‐derived inflammatory cytokines (IL‐6, TNFα) production in TH2 mice. IL‐10 and TNFα secretion were not affected by PTP1B deletion in mice on TH1 responder background but levels of TH1 derived pro‐inflammatory cytokines (IL‐6, INFγ) were reduced. Taken together these data suggest that T cell background might influence BP response to leptin likely by controlling the inflammatory response to leptin.
PDE6s are heterogeneously distributed in native endothelium of mesenteric arteries from mice
Authors: Blanchette Alexandre , Toussaint Fanny , Charbel Chimène , Comtois Philippe , Ledoux Jonathan
Published: June 2021

Cyclic nucleotides (cGMP, cAMP) being key components of cellular pathways, their local levels are the core of important regulatory mechanisms. Although endothelium is the foremost vascular producer of NO, regulatory mechanisms of the NO/cGMP remain to be determined in endothelial cells. Polarity of native endothelial cells also suggests potential cyclic nucleotides microdomains. cGMP‐specific phosphodiesterases, PDE5 and PDE9, have previously been identified in endothelial cells. However, both active isoforms of retina‐restricted PDE6 (α & β) appear to be expressed in native endothelial cells as well. In opposition to PDE5 which is homogeneously distributed in endothelial cells, both PDE6s appear to be localized at the nuclear membrane interface as evidenced by confocal microscopy. However, α isoform is sparsely distributed while β forms a continuous perinuclear ring. Interestingly, PDE6s could not be detected in cultured endothelial cell lines. Dynamic mathematical modeling revealed that the experimental intracellular distributions of PDE6s and PDE5 have a significant impact on local cGMP levels, especially in the perinuclear region where cGMP levels are reduced significantly in response to a pulse of NO. Combined experimental and modeling results suggest that PDE6 might have a significant impact on cGMP‐dependent endothelial function in native arteries. Supported by FRSQ, NSERC and HSFC.
G‐protein Coupled Receptor 30 (GPR30) in the Human Endothelium: New Roles for a Novel Estrogen Receptor
Authors: Chakrabarti Subhadeep , Davidge Sandra T
Published: June 2021

Estrogen is a modulator of endothelial function. Traditionally the effects of estrogen were believed to be mediated through its classical receptors alone. There is increasing evidence of a novel G‐protein coupled receptor GPR30 in mediating some estrogenic actions. While there is evidence of vascular actions of GPR30, this protein has not been identified to date in the human endothelium and its endothelium‐specific roles are poorly understood. We used human umbilical vein endothelial cells (HUVECs) as a model system. A specific pharmacological agonist (G‐1) and an antagonist (G‐15) were used to characterize roles of GPR30. Inflammatory changes were induced by stimulation with the pro‐inflammatory cytokine tumor necrosis factor (TNF). NF‐kappaB activation was determined by degradation of inhibitor kappaB and nuclear translocation of p65. We detected GPR30 protein in HUVECs by both western blotting and immunofluorescence. GPR30 activation by G‐1 increased F‐actin and stress fibre formation in HUVECs which were prevented by G‐15 administration. G‐1 treatment also inhibited TNF‐mediated adhesion molecule expression in these cells, without altering NF‐kappaB activity. Our results suggest that endothelial GPR30 is a novel mediator of actin polymerization and anti‐inflammatory effects downstream of NF‐kappaB.

This work was supported by CIHR.
Temporal changes in connexin 43 (Cx43) expression in pulmonary microvessels of LPS‐treated rat lungs
Authors: Kandasamy Kathirvel , Parthasarathi Kaushik
Published: June 2021

Endothelial Cx43‐containing gap junctions play a major role in the expansion of inflammatory responses in lung. However, it is unknown whether Cx43 expression is modulated during an inflammatory response. To establish this in an LPS‐induced inflammatory model, we instilled LPS (2mg/kg) intratracheally in rats and allowed the animals to recover for either 1, 5, or 14 days. At the end of each recovery period, lungs of the animals were isolated and blood‐perfused. Via a microcatheter introduced through the left atrial cannula, we established blood cell‐free conditions in microvessels. In these microvessels, we determined Cx43 expression by indirect immunofluorescence using a confocal imaging system. Fluorescence intensity quantified along the wall of microvessels revealed that endothelial Cx43 expression declined at day 1 post‐LPS treatment (venule 46.2±6.6; capillaries 10.9±1.0, mean±SE, n=3, p<0.001) compared to untreated controls (83.9±8.2; 43.4±3.6), and was lowest at day 5 (22.2±1.9; 4.5±0.5, p<0.001). However, the expression returned to baseline levels at post‐treatment day 14 (97.5±8.1; 54.9±4.9). Thus, the data indicate a steady post‐insult decline in Cx43 expression with time, and a recovery to pre‐insult levels over a longer period. We interpret that mechanisms that initiate recovery from an inflammatory insult may downregulate Cx43 expression in lung microvessels. (NIH HL75503)
The serine‐threonine calcium/calmodulin‐activated phosphatase calcineurin is involved in regulation of store‐operated calcium entry
Authors: Vasauskas Audrey A. , Wang Xiaogan , Chen Hairu , Wu Songwei , Cioffi Donna L.
Published: June 2021

Disruption of the endothelium leads to increased permeability, allowing extravasation of macromolecules and solutes from blood vessels. Cytosolic calcium entry through a calcium‐selective, store‐operated calcium channel (SOC), Isoc, contributes to barrier disruption. The transient receptor canonical (TRPC) protein TRPC4 is part of Isoc channel structure, and TRPC4 is necessary for proper Isoc channel function, possibly through its phosphorylation status. Indeed, phosphorylation status has been implicated as a regulatory mechanism of activity for a number of TRPC and SOC channels. Preliminary results from our laboratory suggest Isoc channel regulation is dependent on dynamic phosphorylation of TRPC4. However, the kinase(s) and phosphatase(s) involved in TRPC4 phosphorylation are unknown. In this study, we sought to determine whether the phosphatase calcineurin (CN) plays a role in SOC entry. Using a Fura 2/AM approach to measure cytosolic calcium in pulmonary arterial endothelial cells (PAECs), we show that a CN inhibitor (CIP) decreases SOC entry following thapsigargin treatment in pulmonary arterial endothelial cells (PAECs). Luciferase assays confirm inhibition of CN activity by CIP in PAECs. As well, Isoc is significantly attenuated in whole‐cell patch clamp studies of PAECs. Together, these data suggest that CN contributes to regulation of endothelial SOC entry. Supported by 5R00HL089361
Loss of cell‐surface sialic acids activates calcium entry in pulmonary endothelial cells
Authors: Cioffi Eugene A. , Crockett Edward S. , Cioffi Donna L.
Published: June 2021

The endothelial glycocalyx is a complex mixture of glycoproteins, glycolipids and proteoglycans which coats the cell surface. Sialic acid is a carbohydrate residue that is typically found at the glycan chain terminus in a α‐2,3‐Gal‐ or a α‐2,6‐Gal/GalNAc‐linked configuration. We have previously demonstrated that pulmonary microvascular endothelial cells (PMVECs) express principally α‐2,3‐linked sialic acids whilst pulmonary artery endothelial cells (PAECs) express both α‐2,3‐ and α‐2,6‐linked sialic acids. Further, treatment with neuraminidase, to cleave cell‐surface sialic acids, leads to endothelial barrier disruption as evidenced by loss of cell‐cell and/or cell‐matrix adhesions. Calcium entry through store‐operated calcium entry channels is a critical event that results in endothelial barrier disruption. However it is unknown whether selective cleavage of cell surface sialic acids leads to calcium entry. Confluent monolayers of PAECs and PMVECs were treated with neuraminidase from Clostridium perfringens and cytosolic calcium levels measured by the ratiometric dye Fura2/AM. Cytosolic calcium increased following addition of neuraminidase in both PAECs and PMVECs. EDTA addition to the extracellular solution reversed this increase. Collectively our data reveal that loss of cell‐surface sialic acids triggers calcium entry in pulmonary endothelial cells. Supported by 5R00HL089361.
Complex interplay between Pannexin 1 cysteine residues and channel regulation
Authors: Lohman Alexander W , Weaver Janelle L , Billaud Marie , Bayliss Doug A , Isakson Brant E
Published: June 2021

Post‐translational modification of proteins by S‐nitrosylation at unique cysteine residues can profoundly affect protein function. We recently demonstrated the presence of pannexin 1 (Panx1) channels in the vasculature and thus investigated whether Panx1 can be S‐nitrosylated and if this modification affects channel activity. Application of S‐nitrosogluathione (GSNO), a nitric oxide (NO) donor, to HEK cells transfected with wild type (WT) Panx1 caused S‐nitrosylation of the protein as detected by the biotin switch assay. To identify which cysteine was being S‐nitrosylated, we performed multiple cysteine to alanine mutations (C40A, C66A, C346A, C426A) in the murine Panx1 gene. Mutation of cysteine 426 blocked S‐nitrosylation of Panx1, indicating this residue as the site of modification. To assess functional effects of Panx1 S‐nitrosylation, we subjected HEK cells transfected with WT or our Panx1 cysteine mutants to whole‐cell patch clamp recordings. Application of GSNO dramatically attenuated the Panx1 current in HEK cells expressing WT Panx1. However, C426A current was still inhibited similar to WT Panx1 current, whereas C40A and C346A Panx1 channel current were not altered by GSNO. Lastly, we measured dye uptake and ATP release to correlate electrophysiology data. This data suggests a complex interplay between cysteine regulation of pore function and NO.
Rap 1a small GTPase alters extracellular matrix (ECM) deposition in response to TGF‐β1
Authors: Jeyaraj Selvi C , Unger Nicholas T , Stewart James A , Chotani Maqsood A
Published: June 2021

Cardiac fibroblasts are essential to cardiac remodeling by regulating ECM production, maturation and degradation. TGF‐β, transforming growth factor beta, can regulate fibroblast differentiation and activation, and therefore tissue fibrosis. Rap1a, a Ras‐like GTPase, has been implicated in cell migration and attachment, however, its role in regulation of ECM has yet to be determined. In this study, we examined the impact of Rap1 deficiency on cardiac ECM remodeling.

Analysis of left ventricular tissue sections from Rap1a deficient heterozygous or knockout (HET or KO) mice revealed significant decreases in total (picrosirius red‐PASR), soluble and insoluble collagen when compared to wild‐type (WT) controls (Table I). Isolated cardiac fibroblasts from heterozygous Rap1a mice showed a blunted collagen response to TGF‐β, suggesting an alteration in the normal pro‐fibrotic response to this cytokine. Genetic ablation of Rap1a shows an impact on regulation of ECM which may lead to alterations in cardiac remodeling and hypertrophic response.
Effect of melatonin and its combination with therapeutic exercise on Doxorubicin‐induced cardiac toxicity
Authors: Lkhagvasuren Tserentogtokh , Lee Seunghoon , Hong Jeong-Hwa , Hong Yonggeun
Published: June 2021

Doxorubicin is one of the most widely used antitumor drugs in worldwide, but it has many other toxic effects to brain, kidney and heart. Importantly, it was demonstrated that it leads cardiac dysfunction. The aim of this study was to investigate the cardiac toxic‐protective effects of pineal hormone melatonin, exercise and combination of melatonin with exercise treatment on doxorubicin‐induced cardiac toxicity. Rats were divided 4 groups and all groups inflamed with doxorubicin (2.5mg/kg, 3 times per week) during two weeks. Rats were treated with melatonin (10mg/kg), exercise and melatonin combined with exercise. The results showed that all treated groups were decreased left ventricular thickness by result of sagittal section of H&E staining (P<0.05) and increased tibia/heart ratio (P<0.05) compared to control group. Furthermore, morphological changes and cardiomyocyte vacuolization were decreased by results of high magnification images. We suggest with these results that melatonin combined with exercise is a potential therapeutics against doxorubicin‐induced cardiac toxicity. We further aim to investigate a cellular and molecular mechanisms of against doxorubicin‐induced cardiac toxicity in the melatonin combined with exercise treatment.

This work was supported by KRIBB Research Initiative Program (KGM03021112), Republic of Korea.
Role of mitochondria in hyperoxia adaptation
Authors: Zhao Huiwen , Haddad Gabriel G
Published: June 2021

Prolonged exposure to hyperoxia generates excessive reactive oxygen species (ROS) and potentially oxidant injury in every organ including lung and brain; however, the mechanisms underlying tissue susceptibility or tolerance to hyperoxia‐induced oxidant stress remain elusive. Through a long‐term laboratory selection over many generations, we have generated Drosophila melanogaster flies that tolerate tremendous oxidant stress (90%–95%O2), a lethal condition to naïve flies. In the current study, we report that these hyperoxia‐selected (SO2A) flies have a significantly longer life span in hyperoxia and paraquat‐induced oxidative stress. Prolonged hyperoxia induced a significant ROS accumulation and an increased expression of oxidative stress markers including lipid peroxidation and protein carbonyl contents in control flies but not in SO2A flies. Enzymatic assays revealed that antioxidant enzyme activity in control flies was similar to that in SO2A flies. Using electron paramagnetic resonance techniques, we observed that SO2A flies displayed a decreased superoxide yield in state 3 respiration in isolated mitochondria in comparison to control flies. Furthermore, the activity of ETC complex I and complex III was also inhibited in SO2A flies. We conclude that decreased ROS accumulation, through modification of mitochondrial ETC complex activity, plays an important role in hyperoxia tolerance.
MJ33, an inhibitor of the phospholipase A2 activity of peroxiredoxin 6, reduces reactive oxygen species production in a model of endotoxin induced lung inflammation
Authors: Chatterjee Shampa , Feinstein Sheldon I , Dodia Chandra , Hong Nankang , Lee Intae , Madesh Muniswamy , Fisher Aron B
Published: June 2021

NADPH oxidases (NOX) are a major source of enzymatically generated reactive oxygen species (ROS) in mammalian cells; of these, Nox2 activation is thought to play a role in the injury associated with lung inflammation. We reported that Nox2 activation and ROS production occurs through peroxiredoxin 6 (Prdx6) and its subsequent PLA2 activity. Thus blocking Prdx6 PLA2 activity can be a strategy to inhibit ROS generation during inflammation. Here we used MJ33, a potent Prdx6 PLA2 inhibitor and evaluated ROS generation in endothelial cells in vitro and in mouse lungs in vivo post LPS treatment. LPS was either added to endothelial cells in culture or injected i.v. or i.t in mice, while MJ33 was added directly to cell cultures or administered i.t. at doses between 1–10 mole % in liposomes. ROS generation in endothelial cells (monitored by H2DCF fluorescence) showed a 3‐ fold increase after LPS treatment as compared to controls; however addition of MJ33 reduced ROS levels to control values. Lungs from mice treated with LPS showed an almost 10 fold higher rate of ROS production (as monitored by H2O2 specific dye, amplex red) which was abolished by pretreatment with MJ33. Administration of MJ33 in LPS treated mice reduced ROS levels to values as seen with Prdx6 null and Nox2 null lungs indicating that Prdx6 PLA2 activity is critical to LPS induced ROS production and that MJ33 treatment may provide a favorable outcome during inflammation.