bluuebunny/crossref_metadata_2025 huggingface dataset:
CD‐14 expression in hepatic tissue of horses with laminitis
Authors: McGeachy Matthew , Carlisle Katherine , Garza Frank , Stokes Ashley Michelle
Published: June 2021

Researchers hypothesize that laminitis in horses, due to introduction of bacterial components (i.e. endotoxins) into the systemic circulation from GI disturbance, signal an innate immune/inflammatory response via the Toll‐Like Receptor (TLR) Signaling Pathway. TLR‐4 is predominantly responsible for the response to G‐ bacteria and we found this gene was upregulated 3‐fold in laminitic horses compared to controls. The co‐receptor, Cluster of Differentiation 14 (CD‐14), transports endotoxins TLR‐4. We hypothesize that upregulation of TLR‐4 may correlate with expression of CD‐14.

Equine CD‐14 expression was measured using TaqMan RT‐PCR in liver samples from normal, cases with metabolic disease, and those with naturally acquired or experimentally‐induced laminitis. The primers and probe for equine CD‐14 were designed from the NCBI published equine CD‐14 gene sequences (AF200416, NM 001081927). Data is expressed as ΔΔCT (Critical Temperature units).

CD‐14 decreased 0.3561 fold from that of normal horses. Horses with metabolic predispositions for laminitis had a 9.8758 fold increase in CD‐14 expression. Decreased CD‐14 in laminitic horses may not directly correlate with TLR‐4 up‐regulation; therefore more studies need to be performed to understand the potential role of CD‐14 as it relates to laminitis pathogenesis. Funding by the LSU Equine Health Studies Program.
The ApoA‐I Mimetic, 4F, Modulates the Differentiation and Function of Primary Monocytes
Authors: Datta Geeta , Smythies Leslie , White C. Roger
Published: June 2021

ApoA‐I and its mimetic, 4F, inhibit monocyte chemotaxis/adhesion and macrophage incorporation in atherogenic lesions. In this study, we tested the hypothesis that 4F acts by modulating the differentiation and properties of monocytes. Primary human monocytes were treated with 4F, apoA‐I or vehicle control for 7 days. The resulting monocyte derived macrophages (MDMs) were analyzed by flow cytometry for expression of cell surface markers HLA‐DR, CD86, CD11b, CD11c, CD49d, CD14 and TLR‐4. 4F and apoA‐I treatment decreased the expression of HLA‐DR, CD86, CD11b and CD11c compared to control cells suggesting that they modulated monocyte differentiation. Pre‐treatment of monocytes with 4F and apoA‐I inhibited LPS‐induced MCP‐1 expression by 75% but increased 65‐fold the expression of IL‐10. These anti‐inflammatory responses to 4F were associated with a 50% reduction in the expression of CD14 and TLR‐4. 4F and apoA‐I inhibited the ability of MDMs to phagocytose dextran‐FITC beads. They also reduced the adhesion of monocytes (55% reduction) to HUVECs compared to vehicle treatment. These responses were associated with a 90% reduction in the expression of CD49d, a ligand for the VCAM‐1 receptor. These data provide evidence that 4F induces phenotypic and functional changes in MDMs and suggest that the peptide induces the differentiation of MDMs to an M2b "alternatively activated" phenotype.
NEUTROPHIL MIGRATION IS INFLUENCED BY SUBSTRATE STIFFNESS
Authors: Patel Dipan C. , Oakes Patrick , Tang Jay , Cioffi William , Reichner Jonathan
Published: June 2021

Neutrophils are an essential part of the inflammatory response to tissue injury. They are recruited from the bloodstream by chemical signals and migrate through the extracellular matrix to reach the site of injury. Mechanosensing refers to a cell's ability to sense differences in the mechanical properties of its environment. The current study was designed to determine the effect of substrate stiffness on human neutrophil migration using polyacrylamide gels of physiologically relevant stiffness, coated with fibronectin. FMet‐Leu‐Phe in solution was used as a stimulator of random migration. Cell speed was found to correlate inversely with substrate stiffness. This inverse correlation held true when directed migration was stimulated by a local concentration of FMet‐Leu‐Phe delivered via a micropipette tip. It was also found that neutrophils demonstrated greater persistence of directional movement on substrates of increased stiffness. This demonstrated that neutrophils move greater net distances over time despite their slower speeds on stiffer gels. These findings suggest that human neutrophils respond to mechanical cues in the microenvironment in a manner that affects their migration.
Differential Effect of Leptin on Adipose Tissue‐Derived Stromal Cells and Bone Marrow‐Derived Stromal Cells
Authors: jang hoon , hong seong chul
Published: June 2021

The adipocyte actively gives effect to feedback of surroundings and storing excess energy in the form of triglycerides. These cells secrete leptin that regulate food intake because it acts on central nerve system. Also these cells are used by surgery and plastic surgery. The adipocyte has not ability of proliferation which is formed by differentiation from mesenchymal stem cells (MSCs). Recently the concern and increasing of plastic surgery need more adipocyte, but fat poverty patients were hard under the knife of transplant operation, because who have not surplus fat due to transplant. Therefore we tested differentiation effect of rat MSC by induced media which include various sources each of cholesterol, vegetable fat, animal fat and leptin. we confirmed that leptin stimulate to differentiate into adipocyte by GPDH assay. But several studies demonstrated that leptin inhibit the BMSCs to differentiate into adipocyte. Therefore we test dose response of concentration of leptin for differentiation into adipocyte. As a result, 500ng/ml leptin concentration has the most of effect on differentiation of ATSC and BMSC. These results suggest that ATSCs and BMSCs have ability of differentiation.
Sca‐1 positive pancreatic progenitor cells as a cell transplant therapy for treatment of Diabetes Mellitus
Authors: Samuelson Lisa Lynn , Gerber David
Published: June 2021

Objective

We propose a stem cell antigen 1 (Sca1) positive pancreatic progenitor cell (Sca‐PPC) for the treatment of Diabetes Mellitus.

Methods

Cells were isolated from pancreata of 2 week old mice using mechanical and enzymatic digestion. Pancreatic cells were purified for Sca 1 expression and cultured under select conditions. Cells were analyzed by RT‐PCR and Western blot for transcription factors and protein expression. ELISA assays were performed to determine insulin production by the Sca‐PPC

Results

Sca‐PPC were viable and capable of being maintained in culture through seven passages. RT‐PCR demonstrates that the cells maintained the expression of Preproinsulin I and II, Ngn3, Nkx2.2, Nkx6.1, Ins2 and Ptf1 up to passage number 7. Western blot analysis shows the cells are positive for Insulin receptor alpha, PDX1, Ngn3, and amylase through the same passages. ELISA for insulin production demonstrates a basal production of insulin from the Sca‐PPC in maintenance cultures.

Conclusion

Sca‐1 positive pancreatic progenitor cells demonstrate a pancreatic genotype and phenotype in culture. These cells also demonstrate an ability to produce insulin in culture. Sca‐PPCs offer a potential cell type that can be used for the treatment of diabetes.
Cyclic GMP stimulating actions of a novel fully humanized designer natriuretic peptide, CBB‐NP, in human aortic endothelial cells as compared to the native natriuretic peptides
Authors: Lee Candace Y.W. , Huntley Brenda K , Burnett John C
Published: June 2021

Background

CBB‐NP is a novel fully humanized designer natriuretic peptide (NP) integrating the 17‐amino‐acid ring of human C‐type NP (CNP) with the N‐ and the C‐termini of human B‐type NP (BNP). CBB‐NP exerted natriuretic, diuretic, renal‐preserving and cardiac‐unloading actions in anesthetized dogs. In this investigation, we compared for the first time the cGMP stimulating actions of CBB‐NP vs human atrial NP (ANP), BNP and CNP in human aortic endothelial cells (HAEC).

Methods

CBB‐NP was synthesized (SPKMVQGSGCFGLKLDRIGSMSGLGCKVLRRH with a disulfide bond joining the C residues). HAEC were incubated with ANP, BNP, CNP or CBB‐NP for 10 min (10−6 M). Cyclic GMP was quantified by RIA.

Results

CBB‐NP and CNP increased cGMP (pmol/mL) vs control (0.108±0.020 and 0.174±0.036 vs 0.001±0.001, P<0.01 and <0.001, respectively). CBB‐NP elicited a greater cGMP response vs ANP (0.027±0.002, P<0.05), whereas comparable responses were observed vs BNP and CNP. When the native NPs were compared, a greater cGMP stimulating response was detected with CNP vs ANP and BNP (P<0.001 and <0.01, respectively).

Conclusions

The novel designer NP, CBB‐NP, stimulates cGMP in human aortic endothelial cells to a comparable extent as compared to the native BNP and CNP. Moreover, its cGMP stimulating action is greater than that of ANP. The therapeutic potential of CBB‐NP in models of cardiovascular diseases warrants further investigation.

Supported by NIH, HFSA and ASCPT
Resveratrol reverses estrogen‐induced hypertension in female rats
Authors: Subramanian Madhan , Garver Hannah , Fink Gregory D. , Mohankumar Sheba , Mohankumar P. S.
Published: June 2021

Estrogens used in hormone replacement therapy (HRT) are believed to protect against osteoporosis, memory loss, etc. However, recent evidence indicates that HRT increases the risk of coronary heart disease and stroke. Studies from our laboratory suggest that chronic estrogen exposure causes oxidative stress. Since oxidative stress is known to play an important role in the development of hypertension, we wanted to study if chronic exposure to low levels of estrogen causes hypertension in rats. An antioxidant, resveratrol was used to block the effect of estrogen exposure. Adult female Sprague‐Dawley rats were either sham implanted or implanted (s.c.) with 90‐day slow release estradiol‐17 β (E2; 20 ng/day) pellets. Seven weeks later, they were further divided into two groups and fed chow or chow that contained 0.84g resveratrol/kg. Three weeks later, rats were implanted with telemeters via the femoral artery to monitor blood pressure. E2 exposure increased both mean systolic (MSP) and diastolic pressures (MDP) (mm of Hg; mean ± S.E.) significantly to 134.4± 0.4 and 92.8± 0.2 respectively, compared to control rats (124.1±0.5 and 84.8± 0.3 respectively) (p<0.05). While treatment with resveratrol alone did not alter MSP (122.7±0.4) or MDP (85.1±0.2), it was capable of reversing the increase in both MSP (126.5±0.3) and MDP (84.6± 0.2) caused by E2 exposure (p<0.05). These results indicate that chronic exposure to low levels of E2 can increase blood pressure in female rats and this effect is most probably mediated through oxidative stress pathways. Supported by NIH AG 027697.
O2‐ Interacts with Pertussis Toxin‐sensitive G‐proteins to Disrupt α2 Adrenergic Receptor Function in Sympathetic Nerves Supplying Mesenteric Arteries in DOCA‐salt Hypertension
Authors: Dong Hua , Demel Stacie , Fink Gregory , Galligan James , Swain Greg
Published: June 2021

Norepinephrine (NE) is released from sympathetic nerves supplying arteries and veins. We studied differences in NE release mechanisms in vitro in mesenteric arteries (MA) from normotensive (sham) and DOCA‐salt hypertensive rats using amperometry with a microelectrode. NE oxidation currents were larger in DOCA‐salt MA compared to sham MA. Idazoxan (1 µM, blocks α2 adrenergic receptors, α2ARs) increased NE current in sham but not DOCA‐salt MA indicating impaired function of α2ARs in DOCA‐salt rats. α2ARs on sympathetic nerve terminals couple to inhibition of NE release via pertussis toxin inhibited G‐proteins. After 2h incubation in pertussis toxin (2 µg/ml), NE currents increased in sham MA but not DOCA‐salt MA indicating that pertussis toxin sensitive G‐proteins were inactive in DOCA‐salt MA. O2− levels are elevated in MA of DOCA‐salt rats. Therefore, we treated DOCA‐salt rats with the NADPH oxidase inhibitor, apocynin (1.5 mM) in the drinking water for 4 weeks. NE currents were similar in sham MA and apocynin‐treated DOCA‐salt MA and α2AR function was restored in MA from apocynin‐treated DOCA‐salt rats. We conclude that α2AR receptor function is impaired in sympathetic nerves supplying MA in DOCA‐salt rats. This effect is mediated by O2− induced inhibition of PTX sensitive G‐proteins linking α2ARs to NE release. These data support a new mechanism for the beneficial effect of antioxidant treatment in hypertension.
Pharmacological characterization of the serotonin receptor mediating contraction in the rat jugular vein
Authors: Linder A. Elizabeth , Gaskell Geri , Watts Stephanie W
Published: June 2021

The purpose of this study was to characterize pharmacologically the serotonin (5‐hydroxytryptamine; 5‐HT) receptors mediating contraction in the rat jugular vein (RJV). We hypothesized that 5‐HT2A receptor is not the only receptor mediating contraction in RJV. Endothelium‐intact rings of RJV were placed in a tissue bath for measurement of isometric contractile force induced by cumulative addition of agonists (1nM to 10 μM). The contractions induced by the 5‐HT1A/1B and 5‐HT1A receptor agonists, RU 24969 (pD2=‐log EC50 [M]=6.4±0.05) and 8‐OH‐DPAT (pD2=5.5±0.07), respectively, were not as robust as that induced by 5‐HT (pD2=6.8±0.17). The 5‐HT1B/1D/1F agonist sumatriptan failed to induce contraction. The 5‐HT2A receptor agonist DOI (pD2=7.78±0.12) was more potent, whereas the 5‐HT2B receptor agonist BW723C86 was less potent (pD2=6.17±0.12) when compared to the contraction induced by 5‐HT. The antagonists with high affinity for the 5‐HT2A and 5‐HT2B receptors, ketanserin (30nM) and LY266097 (10nM), respectively, caused a right‐shift of 5‐HT‐induced contraction. The 5‐HT1B/1D receptor antagonist GR127935 (10nM) had no effect on 5‐HT‐induced contraction. We conclude that 5‐HT‐induced contraction of RJV is mediated primarily by 5‐HT2A receptor. The involvement of 5‐HT2B and 5‐HT1A receptor subtypes can not be ruled out and deserve further investigation. By identifying receptor mechanisms, we can better understand how the newly discovered serotonergic system in veins may locally modify contraction.
Intracellular calcium silences L‐Type Ca2+ channels in rat small mesenteric veins
Authors: Thakali Keshari , Jordan Renee , Rusch Nancy J
Published: June 2021

Calcium channel blockers (CCBs) exert their antihypertensive effect by reducing cardiac afterload but not preload, suggesting that L‐type Ca2+ (CaL) channels contribute to arterial tone but are silent in resistance veins. Using rat small mesenteric arteries (MA) and veins (MV) perfused at their physiological pressures, we confirmed that the CCB nifedipine prevents KCl‐induced constriction of MA but not MV. We hypothesized that MVs rely on intracellular Ca2+ ([Ca]i) rather than CaL channels for constriction and that [Ca]i silences CaL channels in MVs. Indeed, the SERCA inhibitor, thapsigargin (1 μM), constricted MV but not MA (88±2% vs 3±3% relative to 60mM KCl constriction, respectively), suggesting that SR Ca2+ may fuel MV constriction. When [Ca]i was depleted by thapsigargin and extracellular Ca2+ ([Ca]o) omitted, KCl failed to constrict MVs. After depletion of [Ca]i by thapsigargin in MV and subsequent restoration of 1.5 mM [Ca]o, KCl (60 mM) contractions were partially (61±1%) restored but these contractions were blocked (61%) by 1 μM nifedipine. These data suggest: 1) a link between SR Ca2+ and constriction in MV and 2) that depletion of [Ca]i in MV disinhibits CaL channels, restoring CaL‐mediated Ca2+ influx as a source of venoconstriction. Thus, drugs that selectively deplete venous [Ca]i may represent novel therapies for the treatment of hypertension and other diseases that exhibit elevated venous tone.
Vasculogenesis, a story of glycome and transcriptomal regulation
Authors: Harfouche Rania , Hentschel Dirk M. , Piecewicz Stephanie M. , Basu Sudipta , Print Cristin , Eavarone David , Kiziltepe Tanyel , Sasisekharan Ram , Sengupta Shiladitya
Published: June 2021

Impaired vascularization is implicated in diverse pathological conditions such as cardiac ischemia. Therapeutic vasculogenesis is an emerging concept that can be harnessed to treat this condition, contingent upon better molecular understanding. We evaluated the contribution of the microenvironment during vasculogenesis by modifying heparan sulfate gycosaminoglycans (HSGAGs) using a pharmacologic‐genetic approach. Using an embryoid body model, we show that the de novo development of endothelial cells correlates with an increase in the HSGAG‐sulfating enzyme N‐deacetylase‐N‐sulfotransferase‐1 (NDST1) and that inhibiting HSGAGs blocked vasculogenesis. Similarly, in zebrafish embryos, NDST1 knock‐down resulted in a significantly impaired vasculature. To explore the cross‐talk between the glycocalyx and the cellular transcriptome during vasculogenesis, we identified gene expression patterns that are distinct between wild‐type and NDST1‐knock down zebrafish embryos at stages critical for vasculogenesis. Our analyses revealed an emerging picture of a cascade of pathways downstream of the glycocalyx that are intricately connected to cell signaling, matrix, cell survival/apoptosis and cell fate determination.
Stroke related alterations in the modulation of cerebrovascular pressure dependent constriction (PDC) by nitric oxide synthases (NOS) in stroke prone spontaneously hypertensive rats (SHRsp)
Authors: Daneshtalab Noriko , Smeda John Stanislaw
Published: June 2021

We studied the modulation of PDC by NOS in middle cerebral arteries (MCAs) of SHRsp in relation to hemorrhagic stroke (HS) using a pressure myograph. Confocal microscopy indicated endothelial and neuronal NOS (eNOS, nNOS) in the endotheliumn, nNOS and inducible NOS (iNOS) in the smooth muscle and nNOS in the adventitia of MCAs. In MCAs from prestroke SHRsp, endothelial removal, or the nonspecific NOS inhibitor Nω‐nitro‐L‐arginine methyl ester (LNAME,100µM) shifted PDC to a 100 mmHg pressure step to constricted levels. Endothelial removal did not alter PDC in the presence of LNAME. In de‐endothelialized MCAs, the selective nNOS inhibitor AAAN (10μM, n‐[(4S)‐4‐amino‐5‐[(2‐aminoetheyl)amino]pentyl]‐N‐nitroguanidine) also shifted PDC to more constricted levels. The effects of AAAN could be duplicated but not amplified by LNAME or the NO scavenger hydroxocobalamine (200μM). The iNOS inhibitor 1400W (1.13μM) did not alter tone or PDC. We believe NO generated by endothelial eNOS (and perhaps nNOS) and nonendothelial nNOS (but not iNOS) shifts the operating range of PDC to more dilated diameters in MCAs from prestroke SHRsp. After HS, MCAs from SHRsp could not elicit PDC and were unreactive to LNAME. Reactivity to the NO donor Na‐nitroprusside was unaltered. The MCAs exhibited the presence of eNOS and nNOS, however the latter enzymes appeared to lack the ability to generate NO.
Chronic L‐NAME Treatment in Rats Resulted in A Significant Increase in the Systemic and Right Ventricular Pressure, But the Pulmonary Arterial Pressure was not Altered.
Authors: Sun Xiaowei , Ku David D.
Published: June 2021

The important role of NO in the modulation of vascular reactivity and pressure development is well established. We recently found that an acute and short‐term (1 to 2 weeks) treatment with L‐NAME resulted in a marked systemic hypertension, while the right ventricular (RV) and pulmonary pressure (PP) was not affected, suggesting a marked regional difference in NO vasomodulatory action. Continued L‐NAME treatment for 3 to 4 weeks resulted in a marked left ventricular hypertrophy (LVH), a decreased LV ejection fraction as well as an increase in the RV pressure, while the PP remains protected, suggesting a presence of pressure gradient between the RV and the pulmonary circulation. Using 2‐dimension, M‐mode and Doppler echocardiography we found that chronic NO inhibition resulted in a thickened RV wall, a turbulent RV flow pattern, a decreased RV ejection fraction, and a narrowed area in the RV outflow tract. Further histomorphological studies identified that impediment to RV outflow tract was due to massive hypertrophy of the crista supraventricularis. A similar clinical syndrome of subpulmonary stenosis induced RV outflow tract obstruction has been reported in certain patients with pulmonary disorder. Our findings suggest that this animal model of chronic L‐NAME treatment may be useful to investigate the development of hypertrophic obstructive cardiomyopathy and its clinical management.
Increased production of 20‐HETE contributes to the effects of COX inhibitors to prevent the decrease in lipid peroxidation and increase in catalase activity during endotoxemia
Authors: Tunctan Bahar , Korkmaz Belma , Cuez Tuba , Sahan‐Firat Seyhan , Yildirim Hatice , Tamer Lulufer , Buharalioglu Kemal , Falck John , Malik Kafait U
Published: June 2021

Increased production of NO and prostanoids associated with a decrease in formation of 20‐HETE contributes to development of hypotension during endotoxemia which is minimized by COX inhibitor indomethacin. NO and prostanoids are known to decrease lipid peroxidation (LP) and increase activity of antioxidant enzymes. We investigated whether 20‐HETE contributes to the effects of COX‐1 and COX‐2 inhibitors on the changes in LP and catalase activity (CA) in endotoxin (ET)‐treated rats. ET‐induced decrease in systemic and renal 20‐HETE levels was associated with a decrease in malonedialdehyde levels and an increase in CA in kidney, heart, aorta or mesenteric artery. These effects of ET were prevented by COX‐1 inhibitor, piroxicam, and COX‐2 inhibitor, NS‐398, or a synthetetic analogue of 20‐HETE, N‐[20‐hydroxyeicosa‐5(Z),14(Z)‐dienoyl]glycine (5,14‐HEDGE). An antagonist of 20‐HETE, 20‐hydroxyeicosa‐6(Z),15(Z)‐dienoic acid, reversed the above effects of 5,14‐HEDGE. These data suggest that an increase in 20‐HETE production contributes to the effects of COX inhibitors to prevent the decrease in LP and increase in CA during endotoxemia. This study was supported by Mersin Univ (2006‐3, B Tunctan), NIH (19134‐33A1, KU Malik; GM31278, JR Falck) and the Robert A Welch Foundation (GL 625910, JR Falck).
Lysosomal modulation ameliorates Alzheimer‐type pathology: Evidence of protective cleavage of Aβ1‐42 by modulated cathepsins
Authors: Bahr Ben A. , Butler David , Hwang Jeannie , Estick Candice , Nishiyama Akiko , Kwon Robert , Colon Arthur , Charalambides Ana
Published: June 2021

Alzheimer's disease (AD) involves protein accumulation and progressive synaptic deterioration. Cathepsin proteases exhibit up‐regulation in response to accumulating proteins including Aβ1‐42, and they have been implicated in clearance of AD‐related deposits. Lysosomal modulation with Z‐Phe‐Ala‐diazomethylketone (PADK) involves 3‐8‐fold increases in cathepsin B in APPSwInd and APPswe/PS1dE9 mice, while neprilysin, IDE, and α‐secretase were unchanged. The PADK treatment reduced intracellular Aβ staining, decreased soluble Aβx‐42, and reduced extracellular‐enriched Aβ levels in the two AD models. The corresponding increase in Aβ1‐38 peptide as Aβ1‐42 was reduced indicates that intracellular proteolysis in lysosomes can detoxify peptides, likely through cathepsin cleavage. Associated with its effect on Aβ clearance, deficits in synaptic markers were eliminated in both mouse models by the lysosomal modulator. In addition, behavioral deficits were attenuated, reaching performance scores similar to those of age‐matched non‐transgenic mice. These findings indicate that intracellular processing of Aβ by lysosomes can be targeted to effectively offset the disruption of synaptic integrity and brain function. Lysosomal modulators represent an effective strategy for treating AD and other disorders involving pathogenic accumulations.
Novel Pseudopterosin Analog Elicits New Modality for Use in Wound Repair
Authors: Day Daniel R , Jacobs Robert S.
Published: June 2021

The pseudopterosins are known to be anti‐inflammatory agents (Look, et. al. 1986, PNAS, 83, 6238‐6240), eliciting a reduction in neutrophil infiltration in PMA induced mouse ears and more recently, mediators in acceleration of wound healing. A new pseudopterosin semi‐synthetic analog has been developed which resists glycoside cleavage in the cell via a C‐glycoside linkage. This new compound has been used for the study of cell proliferation effects in HUVEC‐C cell lines. Binding studies indicate that this compound binds to adenosine A2a and A3 receptors with an IC50/hill coefficient of 20 μM/1.2 and 10 μM/2.3 respectively in overexpressed HEK‐293 cells (Zhong, et. al. 2008, J.Org.Chem., 73, 7011‐7016). Although an understanding of the direct molecular mechanism of action has been unknown to this point we will report a tentative theory to explain pseudopterosin effects on wound repair. Modulation of inflammation and cell division/angiogenesis can be important factors in accelerating the wound healing response. Data suggests that Pseudopterosin‐A c‐glycoside elicits a 35% increase in cell proliferation with an ED50 of 3.7 μM over 48 hours in treated HUVEC‐C cell lines. We believe that this effect is mediated by modulation of an adenosine receptor which may act to stimulate the expression and release of growth factors, key to cell division (Ethier et al. 1993, Amer. J. of Phys., 265, H131‐H138). An increase in growth factor expression and release can be potentially important in treatment of chronic wounds since high bacterial and protease loads in wound site act to increase the breakdown of endogenous growth factors. This data suggests that pseudopterosin mediated wound repair may follow two routes; a reduction in inflammatory response, and an increase in cell proliferation. We gratefully acknowledge the U.S. Army Medical Research Program (Grant No. W81XWH‐06‐1‐0089) for its support of this research.
Studies of tissue regeneration in a rat bladder model in vivo
Authors: Burmeister David , Aboushwareb Tamer , Andersson Karl‐Erik , Christ George
Published: June 2021

Functional integration of implanted donor tissue/organs into the host is required for the wide spread clinical success of tissue engineering/regenerative medicine technologies. To do so requires animal models that recapitulate organ/tissue development in an adult environment. As a first step in this direction, we conducted multidisciplinary investigations of bladder regeneration in female F344 rats, following removal of 60‐70% of the bladder (i.e., subtotal cystectomy; STC). Micro CT scans revealed a time‐dependent increase in bladder size (from 1‐8 wks post SCT), that was positively correlated with restoration of bladder function in vivo; despite reduced micturition pressures in vivo and diminished contractility of detrusor tissue strips in vitro. Immunohistochemistry revealed abundant c‐kit staining in the detrusor of STC rats and p63 staining in the basal layers of the urothelium. This suggests a role for stem/progenitor cells in muscle regeneration and urothelial proliferation. Innervation of the regenerated bladder was revealed by positive immunostaining to both PGP9.5 and synaptophysin. These initial studies establish the utility of this research approach as a model system for studying de novo tissue regeneration in vivo. The long‐term goal is to leverage the novel insights obtained to enhance the individual's regenerative capacity, and thus, promote maximal native tissue formation in vivo.
Sexually Diergic Hypothalamic‐Pituitary‐Adrenal (HPA) Responses to Single‐Dose Nicotine, Continuous Nicotine by Osmotic Mini‐Pumps, and Nicotine Withdrawal by Single‐Dose Mecamylamine in Rats
Authors: Gentile Natalie E. , McKlveen Jessica M. , Czambel R. Kenneth , Karwoski Tracy E. , Rubin Robert T. , Rhodes Michael E.
Published: June 2021

HPA responses to acute doses of nicotine (NIC) are sexually diergic: Female rats have higher adrenocorticotropic hormone (ACTH) and corticosterone (CORT) responses than males. We determined HPA responses in male and female rats following: 1) single‐doses of NIC, 2) a single‐dose of NIC immediately following continuous NIC for 2 weeks, and 3) NIC withdrawal by single‐dose mecamylamine (MEC). Blood sampling occurred before and after MEC and NIC administrations. ACTH and CORT were determined by RIA.

Male vs. female HPA responses to single‐dose NIC support previous findings: Female ACTH and CORT responses were greater than male responses. The sex difference to single‐dose NIC remained following continuous NIC, and HPA responses in both sexes were significantly lower in magnitude and duration. The sex difference remained following NIC withdrawal by MEC; as well, the HPA responses were significantly higher in magnitude and duration than in the continuous NIC groups. These results suggest that sexually diergic HPA responses to NIC are reduced and transient following continuous NIC administration, but are enhanced and sustained following NIC withdrawal. The present study may have implications for determining associations among stress, habituation of the HPA axis to chronic NIC use, and NIC withdrawal.
Atrial Natriuretic Peptide Stimulates Rat Pancreatic Beta‐Cell Growth
Authors: You Hui , Laychock Suzanne G
Published: June 2021

Natriuretic peptide receptor‐A (NPR‐A) is a guanylyl cyclase‐linked receptor that generates cGMP in response to atrial natriuretic peptide (ANP). Since elevated ANP levels are associated with diabetes mellitus and hyperglycemia, ANP effects on cultured isolated rat pancreatic islets and insulinoma cells (INS‐1e) were determined. Islets and INS‐1e cells expressed NPR‐A mRNA and protein. To determine the chronic effects of ANP, islets were cultured at 5.5 mM glucose with ANP (1 μM) for 7 days, which increased [3H]thymidine incorporation to 200±36% of control (p<0.05); similarly, ANP increased INS‐1e cell [3H]thymidine incorporation to 163±18% of control (p<0.05). 8‐Bromo‐cGMP (0.5 mM) also stimulated islet [3H]thymidine incorporation to 207±15% of control (p<0.001). The mTOR inhibitor rapamycin failed to inhibit ANP‐stimulated [3H]thymidine incorporation. In ANP‐treated INS‐1e cells, the phosphatidylinositol 3‐kinase (PI3K)/Akt/Foxo1a pathway was activated. Phospho‐Akt and phospho‐Foxo1a were increased by 387±77% and 170 ±12% of control (p<0.05), respectively, and this response was blocked by the PI3K inhibitor LY294002. Cyclin D2 mRNA was upregulated in ANP treated islets and INS‐1e cells by 336±71% and 332±36% of control (p<0.05), respectively. The evidence suggests that the mitogenic response stimulated by ANP treatment involves activation of the PI3K/Akt/Foxo1a/cyclin D2 pathway.
Alpha‐7 Nicotinic Acetylcholine Receptor (nAChR) Characteristics on the Acetylcholine Binding Protein
Authors: Nemecz Ákos , Taylor Palmer
Published: June 2021

The cys‐loop family of ligand‐gated ion channels plays an important role in the chemical‐to‐electrical transduction of neuronal signaling. The well‐studied α7‐nAChR may be linked to nicotine addiction, and shows possible genetic linkages to such diseases as Alzheimer's and schizophrenia. The discovery of a homologous soluble snail acetylcholine binding protein (AChBP) provided a structural surrogate of the extracellular ligand‐binding domain; however, the binding protein, despite the requisite ligand properties, shows only 30% residue identity with the nicotinic receptor family. Here we show systematic mutations of the soluble Aplysia californica towards the α7‐nAChR with simultaneous analysis of binding affinities. Key constructs were subsequently characterized using classical α7‐nAChR selective ligands such as α‐bungarotoxin, (+)‐epibatidine, and methyllycaconitine, as well as crystal structures have been obtained for some of the intermediate constructs. (Support: U01‐NS 05846, GM07752‐29)
G Protein‐Coupled Receptor Kinase 4γ (GRK4γ) Interacts with the Alpha Helical Domain of Gαs
Authors: Andresen Bradley T. , Sisk Kay , Keever Lindsay B. , Bastepe Murat , Jackson Edwin K.
Published: June 2021

GRK4γ is constitutively active, and polymorphisms of GRK4γ are linked to clinical hypertension and can induce experimental hypertension. Recent studies demonstrate that GRK4γ interacts with inactive Gαs without interfering with the G βγ complex. To better understand this interaction and GRK4γ function, we investigated the site of interaction between Gαs and GRK4γ utilizing chimeric Gαs/q and Gαs/i1 subunits (only Gαs and Gα13 interact with GRK4γ). Chimeric Gαs/q suggested that GRK4γ interacts with the amino‐terminal portion of Gαs, which was confirmed with the chimeric Gαs/i1. These data indicate that GRK4γ interacts with amino acids 60 ‐ 138 in the helical domain of Gαs. Sequence alignment between the Gα subunits suggested 3 specific points of interaction in the αB helix (Q111, D115, and S119), but after site directed mutagenesis GRK4γ still bound to Gαs. Therefore, the interaction involves multiple amino acids. Since the structure of the helical domain of Gαs that interacts with GRK4γ resembles a hook, much of the helical domain may be involved in interacting with GRK4γ. Binding to the alpha helical domain of Gαs explains how GRK4γ can bind to inactive Gαs and not displace Gβγ as well as account for its constitutive activity. Support was provided by American Health Assistance Foundation and Department of Veterans affairs VISN 15.
Comparison of agonist‐dependent internalization, downregulation and recycling of M1‐M5 receptors in CHO cells
Authors: Thangaraju Arunkumar , Sawyer Gregory William
Published: June 2021

In this investigation, the agonist‐dependent internalization, downregulation and recycling of muscarinic M1‐M5 receptors was characterized in CHO cells. In internalization assays, cells expressing each subtype of muscarinic receptor were incubated with the muscarinic agonist carbachol for various periods of time for up to 240 min. Cells were then used in intact, whole cell binding assays to determine the amount of receptor remaining on the cell surface. Using this approach, we found that M2 and M4 receptors internalize more extensively than M1, M3 and M5 receptors. In downregulation assays, cells expressing each subtype were incubated with carbachol for 24 h prior to performing intact, whole cell binding assays. We found that the rank order of muscarinic receptor downregulation was M1>M4>M5>M3>M2. For receptor recycling, cells were incubated with carbachol for one hour to induce receptor internalization and then treated with benzidyl choline mustard to alkylate receptors remaining on the cell surface. The amount of receptor returning to the cell surface was then determined at various periods of time for up to 90 min using intact, whole cell binding assays. In these experiments, we found that M1, M3 and M5 receptors recycle more extensively than M2 and M4 receptors. Collectively, our data shows that there are subtype‐specific differences in the agonist‐dependent regulation of muscarinic receptors.
Modulation of α2C ‐Adrenergic Receptor Traffic by Molecular Chaperones
Authors: Filipeanu Catalin
Published: June 2021

Cold exposure stimulates a2C‐adrenergic receptor (a2C‐AR) transport to the cell surface leading to exaggerated vasoconstriction observed in Raynaud Phenomenon. To determine the molecular mechanisms underlying this effect, the a2C‐AR interactions with molecular chaperones were investigated by co‐immunoprecipitation in transfected HEK293T cells. The receptor interactions with calnexin and calreticulin were similar at 37oC and at 30oC. In contrast, exposure to 30oC for 18 h decreased the a2C‐AR interactions with BiP/GRP78 (43 ± 11 %, n=3), suggesting that such prolonged interactions at 37oC may delay the receptor transport. Further, low‐temperature decreased a2C‐AR interactions with with β‐COP (32 ± 9%, n=4), indicating that the retrograde traffic also prevented receptor traffic. Overexpression of BiP prevented the cold‐induced enhancement in α2C‐AR traffic, as determined by radio‐ligand binding. These data indicate that low‐temperature stimulates α2C‐AR transport to the cell surface by decreasing the receptor interactions with specific molecular chaperones. Thus, BiP and β‐COP might be useful biomarkers to detect the individuals susceptible to Raynaud Phenomenon. Supported by NIH Grant 2P20RR018766‐06.
ETB receptor activation changes ETB receptor location in venous but not aortic smooth muscle cells
Authors: Tykocki Nathan Roger , Watts Stephanie W.
Published: June 2021

Endothelin (ET‐1) is a 21‐amino acid peptide that acts as a potent endogenous vasoconstrictor, and is produced by vascular endothelial cells. ET‐1 activates two G‐protein coupled receptor subtypes: ETA and ETB. In arteries, ETA receptor stimulation causes contraction and ETB stimulation causes relaxation. Although ETA stimulation also causes contraction in veins, ETB stimulation can cause either contraction or relaxation. Since ETB receptors cause contraction in veins but not arteries, we hypothesize that venous smooth muscle ETB receptors will relocate within cells in ways arterial ETB receptors do not.

Explants of aortic and venous smooth muscle cells (SMC) were cultured from freshly dissected rat thoracic aorta and vena cava. Cells were exposed to vehicle or the ETB receptor agonist Sarafotoxin‐6c (S6c) (100 nM) for 30 minutes prior to immunofluorescent labeling of ETA receptors, ETB receptors, and nuclei. Untreated aortic and venous SMC expressed ETA and ETB receptors, without significant difference in receptor mean fluorescence. In the presence of S6c, mean ETB receptor fluorescence was significantly greater in nuclei of venous SMC when compared to aortic SMC (137.6±9.4 vs. 96.3±11.5). S6c did not alter ETA or ETB whole‐cell fluorescence in aortic and venous SMC. These findings imply a role of activated ETB receptors in S6c‐induced signaling in venous smooth muscle cells.
Functional Signatures of α‐7 Nicotinic Acetylcholine Receptor Positive Allosteric Modulators: Effects on Channel Biophysics and Pharmacology.
Authors: Peri Ravikumar , Randall Andrew , Bowlby Mark , Yeola Sarita , Jow Flora , Lock Yeung‐Wai , Sitzia Fabrizio , He Lan , Lee Yan , Kramer Angela , Dunlop John
Published: June 2021

Activation of neuronal α7 nicotinic acetylcholine receptors by agonists evokes rapidly activating, desensitizing currents, predominantly carried by Na+ and Ca2+ ions. Positive allosteric modulators (PAMs) of α7 nAChRs are devoid of intrinsic agonist activity but enhance α7 currents with or without effects on channel kinetics. Using patch clamp and Ca2+ flux assays in GH4C1 cells heterologoulsly expressing rat α7 receptors we describe the mechanism of action of α7 PAMs and their modulation by changes in temperature and repeat applications. All PAMs enhanced the current amplitude. Additionally the type‐2 PAMs also slowed channel desensitization. We show that PNU‐120596, a type‐2 PAM exhibited reduced potentiation of current amplitude upon repeated applications with no change in the EC50 of activation. PNU‐120596 also "dedesensitizes" receptors fully desensitized by exposure to high concentrations of agonists. NS‐1738, a type‐1 PAM exhibited a modest decrease in maximal current amplitude upon repeated application accompanied with 3‐fold increase in potency. SB‐206553 a type‐1/2 PAM did not exhibit differences in the extent of current amplitude potentiation or pharmacology upon repeated application. Modulatory effects of PAMs, though significant at room temperature, were attenuated near physiological temperatures. Furthermore, PNU‐120596 but not SB‐206553 largely removed the strong inward rectification usually exhibited by α7 nAChR receptor agonists.
Isothiocyanate attenuates ischemia‐induced cell death
Authors: Yao Hang , Wang Juan , Theodorakis Emmanuel A , Haddad Gabriel G
Published: June 2021

4,4′‐diisothio‐cyanostilbene‐2,2′‐disulfonic acid (DIDS) is a stilbene derivative with pleiotropic biological nature. DIDS not only affects the activity of several ion channels and transporters in mammalian cells, but also mediates cell survival and neurodegeneration. Recently, we and others have observed that DIDS could prevent cell damage in both in vivo and in vitro ischemia models. However, the mechanism underlying DIDS protection is not clear. In this study, we examined the role of the isothiocyanate group (SCN) as the functional moiety in the DIDS molecule and its effect on cell death. Using an in vitro ischemia model (IS) developed in our laboratory that uses organotypic brain slices and propidium iodide fluorescence as an assay for cell death, we compared the protective effect of several stilbene derivatives that have different numbers of SCN groups. Our data show that at 400 µM, DIDS and H2DIDS (both have two SCN groups) blocked IS‐induced cell death by 100%, SITS (with one STC group) blocked by about 50%, and DNDS (with no SCN group) had no effect. These results strongly support the idea that the rescue of cell death by DIDS is dependent on the presence of the STC moiety.
Selective functional sympatholysis promotes blood flow distribution to recruited muscle fibers
Authors: Moore Alex W , Segal Steven S
Published: June 2021

Inferior and superior regions of the mouse gluteus maximus muscle (GM) are perfused and innervated by respective gluteal arteries and motor nerves (MN). Brief stimulation (100 Hz for 500 ms) of the inferior MN produces tetanic contraction that initiates rapid (<1 s) onset vasodilation (ROV) within the recruited (inferior) muscle region. ROV ascends into inferior feed arteries but not along arteriolar anastomoses (AA) into the quiescent (superior) muscle region. We tested whether constitutive activity of a‐adrenergic receptors prevents the spread of ROV. In anesthetized male C57BL/6 mice (3 mo, 25 g), the left GM was exposed and superfused with physiological saline (35 °C, pH 7.4) for intravital microscopy. Vessel diameters were measured in each direction along AA at sites 800 μm from the functional border between respective muscle regions. Stimulation of the inferior MN produced muscle contraction and initiated ROV along AA within the recruited region (diameter, rest=32±2 μm; peak=42±3 μm; max=51±1 μm) but not in the quiescent region. Whereas phentolamine superfusion (1 μM) had no effect on resting diameter, ROV increased in the recruited region and spread along AA into the quiescent region (diameter change, 7±1µm; p<0.05, n=5). Thus constitutive activity of α‐adrenergic receptors enables selective functional sympatholysis to promote blood flow to recruited muscle fibers. (HL086483; MU Fellowship)
Functional vasodilation following murine chronic hindlimb ischemia
Authors: Yocum Matthew David , Cardinal Trevor R.
Published: June 2021

Functional hyperemia and arteriolar vasodilation are impaired with chronic ischemia. We sought to examine the impact of chronic ischemia on collateral artery function. For this we used two hindlimb ischemia models to dissect the impact of different repair processes on collateral function. Ligation of the femoral artery increases shear stress in the muscular branch and results in outward remodeling and arteriogenesis. In contrast, resection of the femoral artery proximal to the muscular branch induces blood flow divergence and neutral/inward remodeling along with greater hypoxia and inflammation. On day 14 after each surgery the diameter of the muscular branch was measured using sidestream dark field (SDF) imaging before and after gracilis muscle stimulation. A slight, but not statistically significant, impairment in functional vasodilation was observed in ligated mice (1.69±0.10 fold diameter increase compared to 1.74±0.04 fold diameter increase). Surprisingly, resected mice exhibited enhanced collateral artery functional vasodilation (2.04±0.20 fold diameter increase) but were also refractory to the restoration of resting vascular tone following the cessation of stimulation. Outward remodeling did not significantly impair vascular function, whereas inward/neutral remodeling and tissue hypoxia induced enhanced vasodilation and impaired vascular tone.
TISSUE OXYGENATION AND OXYGEN CONSUMPTION FOLLOWING HEMORRHAGE AND RESUSCITATION USING A HEMOGLOBIN‐BASED OXYGEN CARRIER AND HUMAN SERUM ALBUMIN
Authors: Song Bjorn K , Connery Michael D , Moon‐Massat P. , Pittman Roland N
Published: June 2021

This study compared the effects on O2 transport of two resuscitation fluids: a hemoglobin‐based O2 carrier (HBOC‐201, Biopure Corp., Cambridge, MA), and 5.9% human serum albumin (HSA), a non‐oxygen carrying colloid solution. Intravital microscopic measurements were made on the spinotrapezius muscle of anesthetized, male Sprague‐Dawley rats. Interstitial (ISF) PO2 was measured using phosphorescence quenching microscopy at baseline, post‐hemorrhage and post‐resuscitation time points. Following hemorrhage (40% blood volume) the animal was maintained in this condition for 30 minutes before resuscitation. Local tissue O2 consumption (VO2) was proportional to the rate of decline of PO2 following arrested flow from rapid inflation of a Saran bag placed around the microscope objective. In both groups (HBOC, N=4; HSA, N=4), baseline ISF PO2 (~60 mmHg) decreased significantly during hemorrhage (to ~2 mmHg), and increased after resuscitation (to ~40 mmHg). Significant vasoconstriction and elevation of mean arterial pressure (MAP) was observed with HBOC‐201. Infusion of nitroglycerin (N=2) along with HBOC‐201 diminished arteriolar vasoconstriction, eliminated the rise in MAP, and increased ISF PO2 (to ~55 mmHg). The findings support the hypothesis that vasoconstriction and elevation in MAP by HBOCs is due to nitric oxide scavenging.
Perfusate composition is a determinant of the hydrodynamically relevant endothelial glycocalyx thickness
Authors: Savery Michele D , Damiano Edward R
Published: June 2021

The presence of a hydrodynamically relevant endothelial‐cell surface glycocalyx, ~ 0.5 µm thick, is well established under normal physiological conditions in vivo. It has also been shown that no hydrodynamically relevant layer exists under standard culture conditions in vitro. The endothelial‐cell surface glycocalyx layer is a highly hydrated structure that is constantly bathed in blood in vivo. It is proposed that there is a constant exchange of molecules between the blood and the glycocalyx that is essential to the integrity of the layer. The aim of this study is to test the effect of various perfusate compositions on the structure of the endothelial glycocalyx in vivo. A micromanipulator is used to introduce a micropipette into the lumen of one of the primary feed arterioles to the mouse cremaster‐muscle. The micropipette is then used to steadily perfuse the microvascular network fed by that arteriole. Micro‐particle image velocimetry data are collected from downstream microvessels and velocity profiles extracted from the data are used to estimate the hydrodynamically relevant thickness of the glycocalyx. Preliminary data indicate that vessels perfused with saline in vivo have no detectable hydrodynamically relevant layer. Data will be presented from vessels perfused with saline, cell‐culture media containing 20% serum, or plasma. Supported by NIH grant R01 HL76499.
Cholesterol feedback: A tale of two membrane proteins and two sterol sensors
Authors: Goldstein Joseph L. , Brown Michael S.
Published: June 2021

Cholesterol is an essential component of animal cell membranes, and its concentration is tightly controlled by a feedback system that operates primarily at the transcriptional level. The feedback system consists of a pair of membrane‐bound proteins, Scap and Insig, that directly bind sterols and sense their concentrations in the membranes of the endoplasmic reticulum (ER). When sterol levels are low, Scap exists in a conformation that allows it to transport membrane‐bound SREBPs from the ER to the Golgi. In the Golgi, sequential proteolysis of SREBP by two membrane‐bound proteases releases the N‐terminal half of the SREBP, allowing its entry into the nucleus where it functions as a transcription factor, activating the genes encoding proteins involved in the synthesis and uptake of cholesterol. When excess sterols build up in ER membranes, Scap binds cholesterol and Insig binds oxysterols. As a result of these binding events, both Scap and Insig undergo conformation changes that result in a series of molecular events that block Scap's ability to transport SREBPs to the Golgi, thus terminating cholesterol synthesis and uptake.