bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of FXR Deficiency in Acute Phase Response After Bacterial Infection
Authors: Chung Sammi , Armstrong Laura , Kong Bo , Guo Grace L.
Published: June 2021

Farnesoid X receptor (FXR) is a ligand‐activated nuclear receptor and a transcription factor, regulating bile acid synthesis and transport. FXR is highly expressed in the liver, and more recently FXR has been shown to play a key role in modulating inflammation. Studies have shown that FXR, when activated, acts as a protective agent against liver inflammation and potentially liver diseases such as nonalcoholic steatohepatitis, a part of fatty liver disease. Furthermore, FXR may regulate acute phase proteins, which are secreted as a result of acute phase response (APR)—a systemic reaction that is important for innate immunity and is caused by infection, trauma, or tissue injury. However, the exact mechanism is not well‐established. Thus, the purpose of this study was to investigate the role of FXR in the regulation of the hepatic APR using a bacterial infection model. To understand the mechanism and the effects, hepatocyte‐specific FXR knockout mice (FXRhep −/−) and wild‐type control mice (WT) were injected with Escherichia coli (E. coli) intraperitoneally. Serum, liver, and spleen were isolated 24 hrs following bacterial exposure, and in vivo bacterial counts were determined in whole blood, liver, and spleen. Gene expression of inflammatory cytokines (Tnfα, Il‐1β, Il‐6,) and acute phase proteins (LCN2, Mcp‐1, and Saa3) were measured using quantitative PCR (qPCR) in both liver and spleen. Compared to WT mice, FXRhep −/− mice had significantly increased bacterial load in the liver and spleen following the E.coli treatment, suggesting that they were more susceptible to bacterial infection; however, there was no significant increase in bacterial load in the blood. Serum activities of ALT and AST in FXRhep −/− mice were also elevated, indicating increased liver damage after E.coli treatment. In addition, following the E.coli treatment, gene expression showed a diminished induction of acute phase proteins, Mcp‐1 and Saa3, along with cytokines IL‐1β, IL‐6, and Tnfα in FXRhep −/− mice compared to WT mice. Furthermore, this infection resulted in high levels of LCN2 induction for both the WT and FXRhep −/− mice. In conclusion, the study demonstrates that FXR might be a putative regulator of hepatic acute phase proteins.

Support or Funding Information

R25ES020721, R01GM104037 and ASPET SURF Intern Program

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization and Evaluation of Sunitinib and Janus Kinase Inhibitors in a Renal Cell Carcinoma Cell Line, Caki‐2
Authors: Budd Kaitlin Mary , Hege Katie , LePage Bernie , Walker Zachary , Wilson George , Hafron Jason , Banes‐Berceli Amy
Published: June 2021

Kidney cancer is one of the leading cancers detected in middle‐aged men and women. The clear‐cell form of renal cell carcinoma (RCC) is the deadliest form of urogenital cancer; 5‐year prognosis once metastasis has occurred is only 5%. The main challenge of RCC diagnosis is that most symptoms of kidney cancer only appear after it has advanced to metastasis, the most common subtype being clear cell renal carcinoma (ccRCC). At this point, the cancer may have already gained resistance to current treatments. One intracellular signaling pathway, the Janus kinase 2 and signal transducers and activator of transcription 1 and 3 (JAK2/STAT1&3) pathway, has already been implicated in multiple other cancers and is involved in perpetuating many hallmarks of cancer including evasion of apoptosis, angiogenesis, tissue invasion, and metastasis. Microtubule associated protein 7 (MAP7) is a protein involved in tubulin and microtubule stability. High MAP7 expression has been correlated with poor prognosis in colon cancer. Additionally, MAP7 has been shown to be involved in chemo resistance via a STAT1 mechanism. Therefore, we hypothesized that increased MAP7 expression via increased JAK/STAT signaling is a cause of increased chemo resistance and cell proliferation in RCC. Therefore, inhibition of this pathway should increase efficacy of current chemotherapies. Available drugs demonstrate different molecular and physical reactions in RCC cells such as those that effect the cancer promoting unregulated JAK/STAT pathway. MTT analysis of the commercial ccRCC cell line, Caki‐2, with a current chemotherapy drug, sunitinib, shows increased cell viability prior to reaching its EC50 value at 24, 48, and 72 hours of 30 uM, 16.5 uM, and 12 uM, respectively, suggesting therapy resistant mechanisms are taking place. When the known JAK inhibitor Zerumbone was evaluated in Caki‐2 via MTT analysis, EC50 values were 175 uM, 32 uM, and 15 uM at 24, 48, and 72 hours, respectively, with increased cell viability observed only in the 24 hour assay. When these two drugs are used in combination with Caki‐2, MTT data suggests resistance to sunitinib and zerumbone exists in Caki‐2 and co‐treatment aided the ability to decrease cell viability by sunitinib, but not Zerumbone. Based on these results, more studies must be completed to determine whether the inhibition of JAK would be a viable and effective option in treatment of RCC. However, preliminary evaluation of Zerumbone in combination with sunitinib has not proven to be effective.

Support or Funding Information

Oakland University Center For Biomedical Research, Zafarna Grant, Oakland University Honors College

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
K145, a sphingosine kinase 2 inhibitor, inhibits solitary plasmacytoma cell growth
Authors: Li Jessica , Li Xiao Jiaoyang , Zhao Derrick , Wang Xuan , Liu Runping
Published: June 2021

Solitary plasmacytoma (SP) is a rare disease. It is characterized by a localized proliferation of neoplastic monoclonal plasma cells either in the bone (solitary bone plasmacytoma, SBP) or in soft tissue (solitary extramedullary plasmacytoma, SEP). The prognosis of SBP is poorer than SEP due to its higher rate of progression to multiple myeloma (MM). There is no uniform consensus regarding the disease prognosis and treatment due to a very limited understanding of the disease pathophysiology. Sphingosine 1‐phosphate (S1P) has been identified as an important signaling molecule promoting various cancer cell growth. S1P is synthesized by two protein kinases, sphingosine kinase 1 and 2 (SphK1 and SphK2, in cytosol and nuclear respectively). Extensive studies have been done with SphK1 in various cancer cells. However, the contribution of SphK2‐mediated signaling in cancer cell growth remains largely unknown, especially in SP. The current study is aimed to examine the expression of SphK2 in SP cells and further define the role of SphK2 in cell growth.

Methods

Studies were conducted in the human SP cell lines, AMO‐1, NCI‐H929 and RPMI8226. The expression of SphK1 and Sphk2 was determined by real‐time RT‐PCR and Western blot analysis and confirmed by DNA sequencing. The SphK2 inhibitor, K145, and gene‐specific shRNA were used to inhibit SphK2 activation and expression. Cell proliferation was determined using a CCK‐8 kit. Cell apoptosis was analyzed by Annexin V‐FITC/PI staining followed by flow cytometry.

Results

The expression level of SphK2 in all tested SP cell lines was significantly higher than SphK1. S1P significantly induced cell proliferation, migration and invasion of SP cells, which was inhibited by K145 as well as gene specific shRNA targeting SphK2. AMO‐1 has the highest SphK2 expression level among all three tested cell lines both at the mRNA and protein levels. The SphK2 expression level is correlated to invasiveness of SP cell growth and inversely related to the sensitivity to K145‐induced apoptosis.

Discussion/Conclusion

SphK1 and SphK2 are all expressed in human SP cancer cells. However, SphK2 expression level is much higher than SphK1. In this study, by using a chemical inhibitor and gene‐specific shRNA, we were able to show that SphK2 plays a critical role in SP cell growth. Due to the limited efficacy of conventional chemotherapy, there is an urgent need to develop new therapeutics. The results in this study suggest that targeting SphK2 may represent a novel therapy for SP.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A potent and selective allosteric PTP4A3 phosphatase inhibitor enhances microvascular barrier function and inhibits human tumor cell growth.
Authors: Lazo John Stephen , McQueeney Kelley E. , Salamoun Joseph M. , Barabutis Nektarios , Catravas John D. , Wipf Peter
Published: June 2021

The metastasis‐associated protein tyrosine phosphatase PTP4A3 is highly expressed in both tumor and endothelial cells. Deletion of the Ptp4a3 gene results in tumors with a decreased vascularity. PTP4A phosphatases appear to regulate several key malignant processes, such as invasion, migration, and angiogenesis, suggesting a pivotal regulatory role in cancer and endothelial signaling pathways. We have identified a potent, selective, reversible, and noncompetitive PTP4A3 inhibitor, 7‐imino‐2‐phenylthieno[3,2‐c]pyridine‐4,6(5H,7H)‐dione or JMS‐053, which markedly enhanced microvascular barrier function after exposure of endothelial cells to vascular endothelial growth factor or lipopolysaccharide. JMS‐053 also blocked the concomitant increase in RhoA activation and loss of Rac1. In human cancer cells, JMS‐053 impeded migration, disrupted spheroid growth, and decreased RhoA activity. These data demonstrate that PTP4A3 phosphatase can be targeted in both endothelial and ovarian cancer cells, and further confirm that RhoA signaling cascades are regulated by the PTP4A3.

Support or Funding Information

The work was supported by grants from the National Institutes of Health (R21 CA191944, R01 CA207288, S10 OD021723, and F31 CA196062), the Fiske Drug Discovery Fund, the Owens Foundation, 4‐VA Collaborative Research Grant, the Cure Alzheimer's Fund, and the Ivy Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of inhibitors for activated Cdc42‐associated kinase (ACK) to reverse v‐Ras‐induced cancerous phenotype of mammalian cells
Authors: Shepherd Jaleel , Nur‐E‐Kamal Alam , Ahmed Ijaz
Published: June 2021

The superfamily of Ras GTPase has been reported to control different aspects of mammalian cell growth. Our lab demonstrated a possible role of Ras‐Cdc42‐ACK signaling in the survival of v‐Ras‐transformed cells. We also found that ACK‐deficient v‐Ras transformed NIH 3T3 cells undergo apoptosis while the parental NIH 3T3 cells grow normally. We proposed a hypothesis that “ACK‐induced phosphorylation plays a critical role in the survival of Ras‐induced transformed cells”. Based on three‐dimensional structure of ACK, our lab in collaboration with other group screens chemicals to identify potential specific inhibitors for ACK. In this report, I will present computer aided screening protocol and the effect of these inhibitors on growth of v‐Ras‐induced transformed and parental NIH 3T3 cells. I have used software to screen and identify potential inhibitors for ACK. These inhibitors were then used to treat v‐Ras transformed and parental NIH 3T3 cells. The cell growth was monitored by MTT assay. I have further studied the effect of these inhibitors on gene expression using immunostaining, immunoblotting, and polymerase chain reaction. I have identified some compounds that have strong affinity for ACK and inhibit growth of v‐Ras transformed cells. Treatment of v‐Ras cells with these inhibitors was found to activate apoptosis. I have used several markers of apoptosis. These results will be described in this report. We are designing novel compounds with more specific binding to ACK. Development of specific ACK inhibitors will be useful in better understanding the role of ACK in the growth of normal and cancer cells.

Support or Funding Information

NIH‐RISE

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelial Dysfunction as a result of Hypercaloric Intake: Underlying Mechanism in Absence of Hyperglycemia
Authors: Alaaeddine Rana , El‐khateeb Mohamed , Saad Evan I. , Fouad Hosny H. , El‐Sabban Marwan , Plane Frances , El‐Yazbi Ahmed F.
Published: June 2021

Endothelial dysfunction is a common occurrence in diabetes, secondary to hyperglycemia. However, little is known about the mechanism underlying the early endothelial damage occurring in the course of diabetes development prior to impaired glycemic control. We used a high‐calorie (HC) diet rat model with delayed development of hyperglycemia to address this question. Aortic rings from HC rats exhibited impaired endothelium‐dependent relaxation of phenylephrine (PE)‐induced tone to acetylcholine (ACh), where higher concentrations of ACh caused an increase in contractile tone. On the other hand, similar relaxation patterns to the exogenous nitric oxide (NO) donor, sodium nitroprusside, were observed indicating that the defect is endothelial in origin. Hence, the possible role of an endothelium‐derived contractile agent, such as endothelin‐1 and 20‐HETE, or an increased production of reactive oxygen species due to uncoupling of endothelial NO synthase, as the basis of increased tone caused by higher ACh concentrations was ruled out. In aortic rings from control rats, endothelium‐dependent relaxation to ACh was comprised of a major NO component with a lesser contribution from inward rectifier potassium channel (Kir)‐dependent mechanisms, particularly at higher ACh concentrations. While both components of the response were affected, HC‐feeding appears to abolish the Kir‐dependent pathway completely. Protein levels of Kir 2.1 subunit were down‐regulated in aortic tissues from HC‐fed rats. Using U‐46619, a thromboxane analogue, to evoke contraction involving calcium sensitization, rather than depolarization, aortic rings from control rats demonstrated a NO‐dependent, hyperpolarization‐independent pattern of relaxation in response to ACh. Despite a slightly more blunted response to ACh, HC rings pre‐contracted with U‐46619 did not show the terminal contraction observed in tissues contracted with PE. These results potentially implicate impaired myoendothelial coupling as the underlying mechanism for endothelial dysfunction in diabetes. Further examination of the role and expression of gap junctions in these tissues in under way.

Support or Funding Information

Supported by AUB MPP fund #320148

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Resveratrol on Contractions of the Rat Tail Artery: Role of the Endothelium
Authors: Peuler Jacob D. , VanAntwerp Ian R. , Phelps Laura E.
Published: June 2021

Our aims were to determine 1) if chemical removal of the endothelium from rat tail arterial tissue was less likely than mechanical removal to compromise long‐term stability of phenylephrine (PE)‐induced contractions of the smooth muscle, and 2) if resveratrol's previously reported abilities to initially transiently enhance and then produce sustained relaxation of such PE‐induced contractions were dependent on the presence of intact endothelium. We found no indication that chemical removal with saponin was more likely than mechanical removal to compromise PE stability, and saponin perfusion was significantly better in regards to the degree of endothelium removal. We also found that after removal of the endothelium using saponin, resveratrol transiently enhanced PE‐induced contractions (average peak time of 8–9 minutes) and then produced sustained relaxations (at the end of 2 hours) in all tissues and to the same degree as observed for all endothelium‐intact tissues. This suggests that endothelium‐independent mechanisms are responsible for resveratrol's initial contraction‐enhancing and sustained relaxing actions. We also discovered a significant correlation between both actions for both endothelium‐intact and endothelium‐removed rings alike such that with greater initial contraction‐enhancing action there is less of the sustained relaxing action. This suggests that the mechanism responsible for resveratrol's contraction‐enhancing action may not be transient at all (even though the action itself is), but rather present over the full duration in which sustained relaxation occurs. Thus, this enhancing mechanism could potentially limit the long‐term ability of resveratrol to lower arterial pressures by way of its relaxing mechanism.

Support or Funding Information

Biomedical Sciences Program of Midwestern University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New Fossil Discoveries from the Miocene Santa Cruz Formation of Argentina
Authors: Perry Jonathan Marcus Glen , Toledo Néstor
Published: June 2021

The Miocene Santa Cruz Formation of Patagonian Argentina yields well preserved skeletons of many groups of mammals, notably platyrrhine primates and sloths – groups known today from only tropical/subtropical environments and occupying purely arboreal niches. The classic high latitude (51 degrees South) Patagonian localities of this formation have been worked by many paleontologists for more than a century. Our project shifts the emphasis from the classical localities of the Lower Member (ELCm) to the comparatively less studied Upper Member (ELAm) farther south. The ELAm was originally reconstructed as a cooler, drier, and more open‐woodland environment. This paleoecological reconstruction is at odds with recent radiometric dates placing the Mid Miocene Climatic Optimum within the ELAm; the new dates imply that the ELAm was generally warmer and wetter than the ELCm. Our recent field expedition focused on three known localities of the ELAm. One of these (Monte Tigre) yielded abundant mammalian remains, including a diverse assemblage of sloths some of which appear to have been semi‐arboreal. There we also collected the first plant macrofossils known from the ELAm (mostly Nothofagus leaf impressions). Another locality (Cañadon Palos) yielded abundant small mammal remains, including the rare marsupial Abderites. The third locality (Cabo Buen Tiempo) yielded sporadic small mammal fossils (mainly rodents and small armadillos) from a tuffaceous sediment. Future work at these localities will include 1. stratigraphic correlation across 25 kilometers of beach cliffs and shelf, 2. continued fossil collecting at the known ELAm localities, 3. prospecting for new ELAm localities, and 4. paleobiological comparisons between ELAm and ELCm mammals focusing on primates and sloths. New fossils from other vertebrate groups and plant remains will permit a richer paleoecological reconstruction of the ELAm than was previously possible.

Support or Funding Information

Generous support for this study was provided by an American Association of Anatomists Fellows Grant to J. Perry.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cymbopogon Proximus Essential Oil Protects Against Isoproterenol Induced‐Cardiac Hypertrophy in Rats
Authors: Althurwi Hassan , Abdel‐kader Maged , Alharthy Khalid , Al‐salkini Mohamad , Albaqami Faisal
Published: June 2021

Cymbopogon proximus (Halfabar), a wild plant that is usually used in folk medicine due to its diuretic and antispasmodic effects, was shown to exhibit several cardiovascular protective actions such as antidiabetic, antihypertensive, antioxidant, and antinflammatory effects. However, no reports are available regarding the investigation of the role of Cymbopogon proximus essential oil on cardiac hypertrophy. Therefore, the goals of the present study were to find whether Cymbopogon proximus could alleviate isoproterenol induced‐cardiac hypertrophy and to determine the underlying molecular mechanisms. For this purpose, male albino rats were treated daily with either Cymbopogon proximus essential oil (800 μl/kg/day, ip), isoproterenol (5 mg/kg/day, ip), or the combination of both. Oil administration was started four days prior to isoproterenol administration and continued concurrently thereafter for another three days. All animal groups were euthanized 24 hours after the last dose of treatment. Thereafter, the hearts were harvested and heart weight to body weight (HW:BW) ratios were measured and sections of hearts were stained with Masson's trichrome and H&E to assess cardiac fibrosis and morphology. Our results showed that isoproterenol alone resulted in a significant increase in HW:BW ratio and marked myocardial fibrosis in treated rats. On the other hand, treatment with Cymbopogon proximus essential oil significantly reduced the isoproterenol‐mediated increase in HW:BW ratio by 55%. However, oil treatment did not affect isoproterenol mediated‐increase in myocardial fibrosis.

Conclusion

Cymbopogon proximus essential oil partly protects against isoproterenol‐induced cardiac hypertrophy with no effect on cardiac fibrosis. These findings provide support for the promising therapeutic effects of Cymbopogon proximus on cardiac diseases.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PREGNANCY RATE OF BUNAJI COWS IN A FIXED TIME AI SYNCHRONIZATION PROTOCOL USING OVATIDE
Authors: Ubah Simon Azubuike , Rekwot Ibrahim Peter , Adewuyi Bode Abdulmujeeb , Omontese Oakina Bobwealth , Adewale Adeyeye Ayodeji
Published: June 2021

This study was aimed to access the reproductive performance of bunaji cows in an ovsynch protocol involving Ovatide. Bunaji cows (n=16) aged 4 – 6 years and weighing between 250 – 350kg were used. Selected cows with average body condition scores (BCS) of 2.5 – 3.5 using 0 – 5 scale from the most emaciated to the fattest. Cows were randomly assigned to 1 or 2 treatment groups for synchronization of ovulation. Treatment group 1 comprising Bunaji (n=8) received 50μg of GnRH (Cystorellin) and 25 mg of PGF2α. While, treatment group 2 comprising Bunaji (n=8) received 50μg of Ovatide and 25 mg of PGF2α. All cows from both treatment groups were inseminated at 16 hours after each second GnRH or Ovatide injections. Cows that showed mucus discharge from the vagina on the day of AI were recorded as well as those that had patent cervix. Cervical dilation was measured by taking note of the cows that had mid cervix insemination due to non‐passage of the AI gun through the cervix. Transrectal palpation was conducted twice at a month interval to select cycling cows within 5 to 12 days of the estrous cycle before initiating Ovsynch protocol. It was repeated on day 45 post – AI to confirm pregnancies in animals. Results showed that the rate of mucus discharge from the vagina and Cervical dilation for Ovatide were 37.5% and 87.5%; and GnRH (Cystorelin) 75% and 87.5% (P > 0.05) and Pregnancy Rates were 0% and 12.5% respectively. There were no significant differences between the two groups. It was concluded that treatment of bunaji cows with 50μg Ovatide in Ovsynh protocol has heat induction potentials zero pregnancy rates. It was recommended that further studies be carried out using graded doses of 50μg, 100μg and 150μg of Ovatide in a fixed time AI synchronization protocol in bunaji cows.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
AMPK Phosphorylation of FXR at Threonine Controls Hepatic Glucose Production
Authors: Han Chang Yeob
Published: June 2021

Farnesoid X receptor (FXR) controls the expression of genes involved in the regulation of energy metabolism. Nonetheless, the regulatory signal of FXR in glucose‐lowering effect and the underlying mechanisms are largely unknown. Based on the functional link between SIRT1 and AMPK, this study explored the effect of AMPK on the phosphorylation of FXR and its role for SIRT1‐mediated hepatic glucose metabolism. SIRT1 overexpression increased the transcriptional activities of FXR response reporter gene, whereas a loss of SIRT1 had the opposite effect. SIRT1‐mediated FXR transactivation was significantly blocked by the inhibition of AMPK. The impact of SIRT1 and AMPK on the regulation of FXR function was strengthened by the pharmacological effect of ajoene. We also found that AMPK directly phosphorylated FXR at Thr156 residue as shown by in vitro kinase, oligopeptide competition and point‐mutation analyses. The overexpression of FXR‐T156A mutant attenuated AMPK‐mediated FXR phosphorylation and the binding of FXR to coactivator. The inhibition of glucose‐6‐phosphatase expression or hepatocyte glucose production by CA‐AMPK or ajoene was abrogated by either FXR knockdown or FXR‐T156A mutant overexpression. Finally, ajoene treatment diminished hyperglycemia and sensitized insulin signaling in high fat diet‐induced obese mice. In conclusion, AMPK phosphorylates FXR at Thr156 downstream of SIRT1, which may contribute to the inhibition of hepatic glucose production.

Support or Funding Information

This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF‐2015R1A2A1A10052663) and the Basic Science Research Program through NRF funded by the Ministry of Education (2017R1D1A1B03028272).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Role of Vascular Endothelial Growth Factor in Hematopoietic Stem Cell Driven Perivascular Niche Remodeling
Authors: Bubenheimer Rebecca K. , Tamplin Owen J.
Published: June 2021

Hematopoiesis is the process that produces the entirety of mature blood cells. A balance between the quiescence and proliferation of hematopoietic stem cells (HSC) is essential to maintain the blood supply. During development, HSCs expand exponentially in an intermediate niche before migration to the adult bone marrow. In zebrafish, the intermediate niche is the caudal hematopoietic tissue (CHT), a vascular plexus in the tail of the embryo, and in mice it is the fetal liver. Previous work by Tamplin et al. 2015 showed that arrival of an HSC triggers endothelial cells (EC) in the CHT niche to remodel and form a pocket around the HSC. Our goal is to uncover the function of the EC pocket and the mechanism of its formation. One candidate for this signaling cascade is vascular endothelial growth factor C (VEGF‐C) and its receptors VEGFR2 and VEGFR3. Within all hematopoietic lineages, VEGF‐C is highly expressed in HSC. VEGFR2/3 are expressed on sinusoidal endothelial cells of the CHT and fetal liver niches. Previous studies have shown a decrease in hematopoietic progenitor numbers when VEGFR3 is knocked out in mouse embryos (Hamada, 2000). Our preliminary data in zebrafish embryos have agreed with these findings. We have seen a decrease in hematopoietic stem and progenitor cells in vivo marked by the transgenic reporter cd41:gfp and in whole mount in situ with c‐myb probe after knocking down VEGFR3 alone as well as in combination with VEGFR2. To further confirm this phenotype, we administered pan VEGFR inhibitor, Semaxinib, and saw a similar decrease in cd41:gfp+ progenitors. We plan to further investigate this phenotype with a variety of small molecule inhibitors, genetic mutants, and synthetic oligo “morpholino” knockdown. Once we establish the mechanism for VEGF signaling in the remodeling of the perivascular EC niche, we plan to investigate the downstream targets. Uncovering the signals that govern the interaction between HSCs and their perivascular niche is clinically applicable in improving the efficiency of HSC transplants.

Support or Funding Information

Department of Pharmacology, Lung and Vascular Biology Training Grant; NIH T32 HL027829

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Complex Interactions of excitatory and inhibitory stimuli in the vascular endothelium
Authors: Ratnayake Manuela , Wilson Calum , Lee Matthew D. , Girkin John M. , Saunter Christopher M. , McCarron John G.
Published: June 2021

The endothelium is the single, innermost, layer of cells in blood vessels and it controls almost all cardiovascular functions. The endothelium regulates immune responses, blood coagulation, angiogenesis, vessel repair, and vascular permeability. To regulate function, the endothelium continuously monitors multiple chemicals that are circulating in the blood or derived from nearby cells (e.g. endocrine, paracrine, autocrine or neurotransmitter signals). These chemicals each provide cues to physiological status. The chemicals are detected and processed selectively by the endothelium to direct resources that adjust physiological function. Some chemicals provide conflicting signals to the endothelium such as those which promote or inhibit vascular tone. How conflicting signals are detected and managed by the endothelium is not understood. The present study was undertaken to determine how the endothelium selectively processes conflicting information. To monitor the behaviour of the endothelium, changes in endothelial cytosolic Ca2+ concentration were measured in ~200 endothelial cells in small mesenteric arteries using the indicator Cal‐520 and high‐resolution imaging. Changes in cytosolic Ca2+ concentration underlies virtually all endothelial functions and so provides a useful measure of endothelial activity. The vasodilator substance acetylcholine evoked Ca2+ increases that initiated in small clusters of cells. As the concentration of acetylcholine increased, the amplitude of the Ca2+ rise and the number of activated cells increased. To examine the interaction of excitatory and inhibitory inputs, acetylcholine (as an excitatory input) at an EC75 (15nM) was used. In the first series of experiments the vasoconstrictor alpha‐adreneric agonist phenylephrine was used. Phenylephrine (10 μM) did not itself evoke any detectable Ca2+ signals in the endothelium. However, phenylephrine inhibited the Ca2+ rise evoked by acetylcholine (15 nM). Phenylephrine inhibited the amplitude of the response in each activated cell and the number of cells activated. By inhibiting endothelial activity, phenylephrine will increase the contractile response operating on the smooth muscle cells. The vasoconstrictor agonist angiotensin (10 μM) did not evoke Ca2+ increases when applied by itself. However, angiotensin increased the amplitude of the Ca2+ response to acetylcholine in each activated cell. The number of cells activated did not change. This effect of angiotensin on the endothelium may serve to limit a vasoconstrictor response. Together these data reveal complex interaction occur between excitatory and inhibitory stimuli acting on the endothelium.

Support or Funding Information

Supported by the Wellcome Trust and British Heart Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alamandine Counteracts Inflammation in the Central Nervous System of Hypertensive Animals.
Authors: Campagnole‐Santos Maria Jose , Souza Lucas AC. , Kangussu Lucas M. , Santos Robson AS. , Bader Michael
Published: June 2021

Alamandine, a new peptide of the renin‐angiotensin system (RAS), has shown similar biological actions of angiotensin‐(1‐7). In SD rats, alamandine produced stimulatory effects when microinjected into medullary areas involved in sympathetic control. On the other hand, lateral ventricle (ICV) infusion of alamandine improved the baroreflex sensitivity of normotensive rats. We investigated the effect of long‐term central increase of alamandine on the arterial pressure (AP) of transgenic TGR(mREN2)27 (TG) hypertensive rats. Twelve weeks old SD and TG rats (Protocol 239/2016, CEUA/UFMG) instrumented with telemetry probes for AP measurement, underwent 28 days of ICV infusion of alamandine (TG+Ala, 200 ng/h) or saline (TG+Sal and SD+Sal; Control; 0.25 μL/h) through osmotic mini‐pumps. The levels of inflammatory cytokines (TNF‐α, IL‐6, IL‐1β, and IL‐10) in hypothalamus and medulla oblongata was determined by ELISA. Long‐term ICV infusion of alamandine produced a small decrease in Mean AP (ΔMAP: −16±8 mmHg, n=5, p<0.05) of TG rats from the first week of treatment, while control rats (TG+Sal) presented an increase in the AP during the same period of time (ΔMAP: +17±7 mmHg, n=5, p<0.05). Alamandine infusion did not alter baseline heart rate or the circadian rhythm of AP or heart rate in TG rats. Further, TG+Sal presented increased levels of TNF‐α, IL‐6, and IL‐1β in the hypothalamus and medulla oblongata. Interestingly, long‐term ICV infusion of alamandine attenuated levels of TNF‐α (SD+Sal: 29.2±3.1 pg/100 mg of protein; TG+Sal: 73.1±2.9 pg/100 mg of protein; and TG+Ala: 49.3±0.4 pg/100 mg of protein; n=4–7, p<0.001) and IL‐6 (SD: 26.8±1.2 pg/100 mg of protein; TG+Sal: 65.5±4.3 pg/100 mg of protein; and TG+Ala: 42.3±1.8 pg/100 mg of protein; n=4–7, p<0.001) in the hypothalamus and the levels of TNF‐α (SD: 23.5±2.0 pg/100 mg of protein; TG+Sal: 56.9±4.5 pg/100 mg of protein; and TG+Ala: 40.2±1.2 pg/100 mg of protein; n=4–7, p<0.01) in the medulla oblongata of TG hypertensive rats. Treatment with alamandine also increased the levels of IL‐10 (SD: 20.0±0.7 pg/100 mg of protein; TG+Sal: 19.2±0.52 pg/100 mg of protein; and TG+Ala: 24.2±1.4 pg/100 mg of protein; n=4–7, p<0.05) in the hypothalamus of TG rats. Our data show that long‐term ICV infusion of alamandine has anti‐inflammatory effect in areas related to cardiovascular control in transgenic hypertensive rats. This effect was associated with a decrease in AP of these animals. Our data suggest alamandine as an alternative route for the RAS in the central control of AP.

Support or Funding Information

FAPEMIG, CNPq, INCT‐Nanobiofar, CAPES/DAAD (PROBRAL), and FAPEMIG/CNPq (Universal Projects to MJC‐S)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of cardiovascular hemodynamics in response to recumbent positions by using magnetic resonance imaging
Authors: Sasaki Konosuke , Ota Hideki , Kimura Tomoyoshi , Onuma Tomoya , Nagasaka Tatsuo , Saiki Yoshikatsu , Maruyama Ryoko
Published: June 2021

Limited research has been performed to clarify hemodynamic response to different positions, namely the supine, left recumbent, and right recumbent positions. In general, magnetic resonance imaging (MRI) has been reported to be more reliable and reproducible technique than an ultrasound scan for accurate assessment of hemodynamics. Our aim was to evaluate whether the left and right recumbent positions affect cardiovascular hemodynamics compared with the supine position by using MRI assessment. Between June 2017 and August 2017, 12 healthy men aged 26.8 ± 3.1 years were recruited in this study. We measured the following parameters in each posture: heart rate (HR), stroke volume index (SI), cardiac index (CI) in the left ventricle using cine‐MRI, and blood flow velocity and blood volume in the superior vena cava (SVC) and inferior vena cava (IVC) using phase‐contrast MRI. After lying in the supine position, the participants maintained each recumbent position in random order at an angle of approximately 45°. The HR in the left recumbent position and SI in the right recumbent position were observed to be the lowest among those in the other positions. In both recumbent positions, CI tended to decrease compared to that in the supine position. In the right recumbent position, blood volume in the SVC and IVC was significantly lower than that in the supine position. These results suggest that a low HR in the left recumbent position and low blood volume in the right recumbent position result in a decrease in CI in both the right and left recumbent positions.

Support or Funding Information

This work was supported in part by a grant from North Japan Academy of Nursing Science.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High fat diet induces early diastolic dysfunction and cardiac hypertrophy in mice
Authors: Utreras‐Mendoza Yildy Priscilla , Peña Juan Pedro , Montesinos Luis , Sánchez Gina , Donoso Paulina
Published: June 2021

Obesity is a major risk factor for the development of cardiovascular diseases including cardiac hypertrophy. Mice fed with a high fat diet (HFD) become obese after 7–8 weeks and eventually develop cardiac hypertrophy. Yet, the time course of the development of the diet‐induced cardiac hypertrophy is largely unknow.

To study the initial events leading to hypertrophy we fed mice (C57BL/6) with a HFD (60% fat, 20% proteins and 20% carbohydrates) and measured cardiac dimension and function by echocardiography‐doppler after 8 and 12 weeks on this diet. We also determined the mRNa expression for hypertrophic markers such as BNP and RCAN1.4 and since obesity induces the expression of NADPH oxidase in several tissues, we also measured the expression of NOX2 and NOX4, the two isoforms of NAPDH oxidase expressed in the heart.

We found that after 8 weeks on a HFD, mice show normal cardiac dimensions but a decrease in the ratio of the peak velocity in early diastole (Early filling: E) to peak velocity flow in late diastole (Active filling: A) (E/A ratio), indicating the presence of diastolic dysfunction. By 12 weeks E/A ratio is normalized (pseudo normalization) but there is a significant increase in the thickness of the interventricular septum, the left ventricular posterior wall and the heart weight/tibia length ratio, all signals of hypertrophy. At the molecular level, other signs of hypertrophy were significant increases in the mRNa expression of BNP and RCAN1.4. We also found an increase in the expression of NOX4 from 8 weeks but NOX2 expression was unchanged. Until 12 weeks, however, there were no signs of oxidative stress such as a decrease in total glutathione concentration or GSH/GSSG ratio or an increase in carbonylated proteins in the heart.

These results suggest that cardiac hypertrophy develops at 12 weeks as a compensatory response to the diastolic dysfunction induced by the HFD. Redox signaling involving NOX4 may underlie this response

Support or Funding Information

Funded by Fondecyt grant 1160704 (PD) and Doctoral fellowship 21160378 (YUM)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Nuclear Receptor NR4A2 Coordinates Transcriptional Remodeling of Metabolic, Calcium, and Growth Signaling Networks in Adult Rat Ventricular Myocytes
Authors: Ashraf Sadia , Harmancey Romain
Published: June 2021

Sustained elevation of sympathetic activity is an important contributor to pathological cardiac hypertrophy, ventricular arrhythmias, and contractile dysfunction in heart failure. Better understanding of β‐adrenergic signaling regulation in cardiomyocytes should provide therapeutic insights for heart failure management. The orphan nuclear receptor NR4A2 is an immediate early response gene activated in the heart under various stress conditions. The goal of this study was to identify the transcriptional networks regulated by NR4A2 in cardiomyocytes and their effect on cellular remodeling induced by sustained β‐adrenergic stimulation. Isoproterenol (ISO; 10 μM) induced a transient increase in NR4A2 mRNA levels in H9c2 cardiomyoblasts (5‐fold) and in isolated adult rat ventricular cardiomyocytes (ARVM; 80‐fold) that peaked after 1 hour before returning to baseline levels within 24 hours of stimulation. In addition, ISO induced rapid and transient nuclear localization of NR4A2 as visualized by immunofluorescence. Total RNA sequencing revealed that adenovirus‐mediated overexpression of NR4A2 in ARVM led to the regulation of genes associated with cAMP signaling, cell growth and proliferation, glucose metabolism, and calcium homeostasis. These transcriptional events were accompanied by the down‐regulation of genes encoding pro‐hypertrophic transcription factors implicated in pathological remodeling of the stressed heart (Nfatc2, Nfatc3, Gata4, Gata6, and Stat5a). In ARVM chronically treated with ISO for 48 hours, NR4A2 overexpression prevented both the up‐regulation of the pathologic hypertrophic marker BNP and an overall increase in protein synthesis rates as determined by measurement of [3H]leucine incorporation. The overexpression of NR4A2 also prevented ISO‐induced increase in cell surface area for both ARVM and H9c2 cardiomyoblasts. In conclusion, the results expose NR4A2 as a potential regulator of cardiomyocyte metabolic and functional remodeling under stress conditions. NR4A2 may promote antihypertrophic pathways to counteract the detrimental effects of excessive sympathetic system activity.

Support or Funding Information

Supported by grants R00 HL112952, P01 HL051971, and P20 GM104357 from the National Institutes of Health

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the synaptic regulation of kisspeptin/neurokinin B/dynorphin (KNDy) neurons in a mouse model of polycystic ovarian syndrome
Authors: Moore Aleisha M. , Coolen Lique M. , Lehman Michael N.
Published: June 2021

Polycystic ovarian syndrome (PCOS) is the most common cause of infertility in women of reproductive age worldwide. Women with PCOS exhibit the neuroendocrine phenotype of increased luteinizing hormone (LH) pulse frequency, which drives the downstream symptoms of anovulation, cyst formation and hyperandrogenism at the ovary. LH hypersecretion is indicative of impaired central steroid hormone feedback to gonadotropin‐releasing hormone (GnRH) neurons, the final output cell in a large network controlling fertility. Steroid hormone feedback control of GnRH neuron activity and GnRH/LH pulsatile release are hypothesized to be regulated by a population of neurons in the arcuate nucleus (ARC) that co‐express the peptides Kisspeptin, Neurokinin B and Dynorphin, commonly known as KNDy neurons. Therefore, we hypothesized that changes in the regulation of KNDy neuron activity could lead to elevated GnRH neuron activity and GnRH/LH hypersecretion in PCOS. To investigate this hypothesis, we used a prenatal androgen‐induced mouse model of PCOS to determine whether synaptic input to KNDy neurons from the major neurotransmitters GABA and glutamate is altered. Pregnant proDynorphin‐Cre/yellow fluorescent protein (pDyn‐Cre/YFP) transgenic mice were injected subcutaneously with dihydrotestosterone or an oil vehicle on days 16, 17 and 18 of pregnancy. Female PNA (n=4) and prenatal vehicle‐treated (PNV, n=4) pDyn‐Cre/YFP offspring were studied at 60–80 days of age during adult life. Triple‐label immunofluorescence was performed in coronal brain sections for YFP, the vesicular glutamate transporter 2 (vGluT2) and the vesicular GABA transporter (vGaT) to enhance YFP in KNDy neurons and visualize glutamatergic and GABAergic terminals, respectively. The soma of twenty KNDy neurons were analyzed per animal using confocal microscopy. PNA treatment had no effect on the number and density of vGluT2 appositions to KNDy neurons, indicating glutamatergic input is not altered in PNA mice. However, the number (p<0.01) and density (p<0.01) of vGaT appositions to KNDy neurons was significantly reduced in PNA mice when compared to controls. These results indicate that PNA treatment reduces GABAergic input to KNDy neurons. As GABA is classically an inhibitory neurotransmitter, this may lead to increased KNDy neuron firing in PNA mice that drives higher GnRH neuron activity and GnRH/LH pulsatile release, replicating the PCOS neuroendocrine phenotype. The location and phenotype of GABAergic neurons with reduced input to KNDy neurons in PNA mice is so far unknown and deserves further study.

Support or Funding Information

Aleisha M Moore is an American Association of Anatomists Scholar and this research was in part funded by the American Association of Anatomists

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proteomic Determination of the Rat Native Inner Medullary Collecting Duct Lysine Acetylome and Phosphoproteome
Authors: Hyndman Kelly Anne , Yang Chin‐Rang , Jung Hyun Jun , Umejiego Ezigbobiara N. , Chou Chung‐Ling , Knepper Mark A.
Published: June 2021

Phosphorylation and lysine (K)‐acetylation are dynamic post‐translational modifications of proteins. K‐acetylation of histone proteins is classically regarded as a critical regulatory pathway for “opening” chromatin structure and promoting transcription. Previous proteomic studies have identified over 170,000 phosphorylation sites, and greater than 15,000 K‐acetylation sites of mammalian proteins. We recently reported that the inner medullary collecting duct (IMCD), which functions in the regulation of vasopressin‐dependent water‐reabsorption, expresses many of the enzymes (acetyltransferases and deacetylases) that can modulate K‐acetylation. The purpose of this study was to determine the K‐acetylated or phosphorylated proteins expressed in IMCD cells. Second, we asked whether vasopressin V2 receptor activation significantly affects the IMCD acetylome or phosphoproteome? K‐acetylated or serine‐, threonine‐, or tyrosine‐phosphorylated peptides were identified from native rat IMCDs using proteomic analysis with four different enzymes (trypsin, chymotrypsin, ASP‐N, or Glu‐C) to generate a high‐resolution proteome. We identified 709 K‐acetylated sites that mapped to 431 unique proteins. Of these K‐acetylated sites 583 (64%) were novel. The K‐acetylated proteins were expressed in all compartments of the cell, and were enriched in pathways including glycolysis (12 proteins), the TCA cycle (8 proteins), and vasopressin‐regulated water reabsorption (8 proteins). In these same IMCD cells, we detected 312 phosphorylated sites that mapped to 215 proteins, including AQP2 peptides that were phosphorylated at 4 serines: 256, 261, 264, and 269. Moreover, we identified 155 novel phosphorylation sites. Acute dDAVP did not significantly affect the IMCD acetylome; however it did significantly affect previously known vasopressin‐regulated phosphorylation sites. In conclusion, presence of K‐acetylated proteins involved in metabolism, ion and water transport in the IMCD points to multiple roles of K‐acetylation beyond its canonical role in transcriptional regulation.

Support or Funding Information

K01‐DK‐105038 to K. A. H., projects ZIA‐HL‐001285 and ZIA‐HL‐006129, M. A. K.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.