bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of cannabidiol or morphine on mechanical sensitivity and place conditioning induced by chemotherapy‐induced peripheral neuropathy in mice
Authors: Safdar Omar , Neelakantan Harshini , Walker Ellen A , Ward Sara Jane
Published: June 2021

Paclitaxel is a chemotherapeutic agent commonly associated with chemotherapy‐induced peripheral neuropathy (CIPN). We have recently shown that the phytocannabinoid cannabidiol (CBD) can prevent the development of paclitaxel‐induced mechanical allodynia in female C57Bl/6 mice. In the present set of studies, we assessed acute effects of morphine (2.5–10 mg/kg) and CBD (2.5–10 mg/kg) in reversing paclitaxel‐induced neuropathic pain using two assays: i) traditional stimulus‐evoked mechanical allodynia; and ii) non‐traditional place‐conditioning assays to measure non‐evoked affective components of neuropathic pain. Results showed dose‐dependent reversal of paclitaxel‐induced mechanical allodynia by morphine and significant reversal by CBD at all the three doses tested. We then studied the effects of paclitaxel on CBD (2.5–10 mg/kg) and morphine (2.5–10 mg/kg)‐induced place conditioning. Results showed that CBD produced significant place‐preference only in mice that were in the paclitaxel‐induced pain state, while morphine was rewarding in both naïve mice and mice experiencing neuropathic pain. Taken together, these results demonstrate that CBD unlike morphine is effective at relieving chemotherapy‐induced neuropathic pain with no associated psycho‐active effects. These assays also support the clinical observation that opioids are not effective in the treatment of neuropathic pain.
HECOGENIN REDUCES HYPERNOCICEPTION IN MICE
Authors: Quintans Jullyana Siqueira , Antoniolli Angelo , Oliveira Makson GB , Santana Michele F , Santana-Filho Valter J , Branco Alexsandro , Almeida Jackson RGS , Taranto Alex G , Barreto Rosana SS , Quintans Lucindo J
Published: June 2021

The present study examined the anti‐hypernociceptive effect of the hecogenin (HG) in mice. For assess this effect were realized models of mechanic hypernociception induced by carrageenan (CG) and Tumor Necrosis Factor‐α (TNF‐α). The mice were pre‐treated with vehicle (saline + Tween 80 0.2%; p.o.) or HG (5, 10 and 20 mg/kg, i.p.). Thirty minutes after the treatments, were injected in region subplantar of the rear right paw: 20 μL of CG (300 μg/paw) or TNF‐α (100 pg/paw). The hypernociception was assessed in times of 0.5, 1, 2 and 3 hours after the injection of CG and TNF‐α, with digital Von Frey. It was observed that HG was able to maintain the baseline nociceptive threshold in two tests. The results suggest anti‐hypernociceptive property of HGN, probably by a mechanism inhibition of production of cytokines, which activate various reactions connected with inflammation, reducing thereby hipernociception.

Financial support: FAPITEC/SE, CAPES, CNPq (Brazil).
Direct Physical Scaffolding of Muscarinic M3 Receptor Signal Transduction Pathways
Authors: Kan Wei , Malik Sundeep , Faibis Guy , Smrcka Alan V.
Published: June 2021

There is growing recognition that G protein‐coupled receptors (GPCRs) can be pre‐coupled to signal transduction partners to serve a specific and efficient signaling function. We previously found that the third intracellular loop (ICL3) of muscarinic M3 receptor (M3R), expressed as a purified protein fused with glutathione‐S‐transferase (GST), bound purified Gαq, Gβ1γ2, and phospholipase Cβ3 (PLCβ3). PLCβ3 binding to full length M3R in intact cells was only partially dependent on amino acids 275–470 of ICL3, suggesting that the M3R‐PLCβ3 binding interface is comprised of multiple contacts. Further definition of the PLCβ3 binding surface on M3R using purified GST fusion constructs confirmed the multiplicity of contacts with PLCβ3. Contacts close to the membrane interface were found at distinct amino and carboxyl terminal regions of ICL3, ICL2, and C terminal tail. This suggested that rather than being simply tethered to M3R, PLCβ binds to a broad intracellular surface of M3R proximal to the Gαq activation site. M3R binding sites were also defined for Gαq and Gβ1γ2, which were distinct from those for PLCβ3 and from sites involved in direct coupling to the GαqGβγ heterotrimer. Mapping and functional characterization of the PLCβ3 binding surface in the intact M3R will be presented. We propose that the observed receptor‐effector direct scaffolding represents an additional level of fine‐tuning GPCR signaling output.
PROTEASE ACTIVATED RECEPTOR 2 (PAR2)‐MEDIATED ACTIVATION OF PROTEIN KINASE A
Authors: Erb Samuel J , Wang Zaijie Jim
Published: June 2021

Mast cell degranulation and evoked protease‐activated receptor 2 (PAR2) activity has been implicated in inflammation and chronic pain. Both protein kinase C and cAMP‐dependent protein kinase (PKA) transduce activated PAR2 signaling. However, it is unclear how PKA is activated by PAR2. In this study, we tested the hypothesis that activated PAR2 modulates the activation of PKA in a temporal manner. Acute treatment of HEK293 cells with PAR2 agonists SLIGRL−NH2 or the small molecule AC264613 resulted in a time and dose dependent inhibition of cAMP accumulation that could be reversed by administering a PAR2 antagonist FSLLRY−NH2. Furthermore, prolonged exposure of PAR2 to its agonists led to a paradoxical time‐dependent upregulation of intracellular cAMP levels. These effects are abrogated by pre‐treatment with pertussis toxin and suggest that PAR2 is coupled to the Gi protein. These results suggest a mechanism for the activation of PKA and affirm PAR2 as a significant regulator of inflammation and pain signaling.
Reactive Oxygen Species are required for β2 adrenergic receptor mediated β‐arrestin signaling
Authors: Singh Monalisa , Moniri Nader H.
Published: June 2021

The β2‐adrenergic receptor (β2AR) is the prototypical G proteincoupled receptor and is well‐known to elicit biological effects through both G protein‐dependent and G protein‐independent signaling cascades. Agonism of β2AR has been shown to promote phosphorylation and activation of extracellular signal‐regulated kinases (ERK1/2) via a G‐protein pathway which transpires rapidly upon receptor agonism, as well as by a distinct G protein‐independent pathway mediated by β‐arrestins, which occurs at later time points. Our laboratory has previously shown that β2AR agonism promotes generation of intracellular reactive oxygen species (ROS), and that β2AR‐associated G protein signaling is dependent on ROS. In this study, we examine the ROS dependency of the β2AR‐β‐arrestin pathway. Using agonist mediated ERK1/2 phosphorylation as a functional endpoint, our results show that inhibition of ROS abrogates both the G protein and β‐arrestin linked pathways which promote ERK1/2 phosphorylation. Importantly, both components were rescued upon exogenous administration of H2O2, suggesting a role for ROS in stabilization of β2AR signaling. Using both coimmunoprecipitation and bioluminescence resonance energy transfer, we also show that ROS are required for direct interaction of β2AR with β‐arrestin. Together, our results show that ROS serve purposeful roles in promoting both G protein‐ and β‐arrestin‐dependent signaling of the β2AR.

This study was funded, in part, by a New Investigator Program grant (to N.H.M.) from the American Association of Colleges of Pharmacy
Implication of reduced levels of intracellular cAMP in enhanced expression of Gi proteins and hyperproliferation of vascular smooth muscle cells from spontaneously hypertensive rats: cellular mechanisms
Authors: Gusan Svetlana , Ananad-Srivastava Madhu
Published: June 2021

We earlier showed that vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibited an increased expression of Gi proteins and hyperproliferation, whereas the levels of cAMP were decreased as compared to WKY control rats. The enhanced levels of endogenous AngII and ET‐1 in VSMC from SHR were shown to contribute to the increased expression of Gi proteins through the transactivation of EGFR and MAP kinase signaling pathways. The present study was undertaken to examine if the decreased levels of intracellular cAMP in VSMC from SHR contribute to the enhanced expression of Gi proteins and hyperproliferation and to explore the underlying signaling pathways. Protein expression was evaluated by Western blot analysis and cell proliferation by thymidine incorporation. Treatment of VSMC from SHR with dbcAMP attenuated the enhanced expression of Gi proteins and the enhanced proliferation to control levels. In addition, the increased phosphorylation of ERK1/2, c‐Src, EGFR and PDGFR in VSMC from SHR was also inhibited by dbcAMP. Furthermore, dbcAMP decreased the enhanced production of superoxide anion and enhanced expression of Nox4, p47 proteins. These results suggest that the decreased levels of intracellular cAMP in VSMC from SHR contribute to the enhanced expression of Gi protein and proliferation through ROS‐mediated EGFR/PDGFR activation and ERK1/2 signaling pathway (Supported by CIHR).
Gravin‐Dependent Multi‐Kinase Signaling Modulates Cardiac Function
Authors: Guillory Ashley Nicole , Tran Paul , Bilal Khadijah , Wijaya Cori , Diaz Andrea Diaz , McConnell Bradley
Published: June 2021

The purpose of this study was to investigate the effect of knockout of gravin, an A kinase anchoring protein, on the regulation of cardiac function by protein kinase A (PKA) and protein kinase C (PKC). We measured PKA activity, PKC activity and PKA substrate phosphorylation in wild‐type (WT) and gravin knockout (KO) mice stimulated with isoproterenol (ISO; 30 mg/kg/day x 14 days). Basal PKA activity was significantly increased in both WT and KO mice treated with ISO (WT Vehicle: 0.023±0.0041; WT ISO: 0.098±0.021; KO Vehicle: 0.020±0.0021; KO ISO: 0.069±0.0090; A.U.; p=0.0023). However, the WT ISO and KO ISO mice had a decreased response to cAMP‐stimulated PKA activity compared to their respective controls (WT Vehicle: 0.44±0.0094; WT ISO: 0.35±0.027; KO Vehicle: 0.46±0.011; KO ISO: 0.35±0.026; A.U.; p=0.0043). Basal PKC activity was significantly increased in untreated KO animals (WT Vehicle: 0.069±0.012; KO Vehicle: 0.13±0.022; A.U.; p=0.041). Additionally, there was a significant increase in phospho‐phospholamban in the KO ISO group compared to both its control and the WT ISO group (p=0.0151). Furthermore, phospho‐troponin I was significantly increased in the WT ISO group compared to both its control and the KO ISO group (p=0.033). These data indicate that the absence of gravin has a profound effect on the activity and action of PKA and PKC. This research was supported by NIH R01HL085487.
Epinephrine is required for the IL‐13 induced increase in inflammatory cells in murine airways
Authors: Thanawala Vaidehi Jatin , Al-Sawalha Nour , Forkuo Gloria Serwaa , Omoluabi Ozozoma , Knoll Brian , Bond Richard
Published: June 2021

Asthma is characterized by chronic inflammation of the lungs, including inflammatory cells migrating to the lungs, mucus production and airway hyper‐responsiveness. We have shown that in human bronchial epithelial cells, epinephrine (Epi) is required for mucus production in response to the inflammatory cytokine IL‐13. We treated mice with IL‐13 to study the role of Epi in the development of the asthma phenotype. Mice were administered reserpine (5 mg/kg i.p.) to pharmacologically deplete Epi. Reserpine treatment produced >99% reduction of adrenal Epi. We administered IL‐13, intra‐tracheally before or after reserpine treatment. IL‐13 treatment produces minimal inflammatory cells in the airways after 24 hours, with peak responses occurring at about 72 hours. Administration of reserpine prior to IL‐13 prevented the increase in inflammatory cells. However, administration of reserpine 24 hours after IL‐13 treatment did not prevent the increase in inflammatory cells. Thus depletion of Epi prior to stimulation with IL‐13 prevents inflammatory cells from reaching the lungs. However, administration of reserpine at a time point where little or no inflammatory cells are in the lungs does not prevent their accumulation following IL‐13 administration. These results suggest that Epi is required for the production or recruitment of inflammatory cells to the lungs.

Supported by NIH 1R01A179236 to RAB
Effects of proteasome inhibition by a novel imidazoline on ovalbumin‐induced airway inflammation and hyperresponsiveness
Authors: Azevedo Lauren , Proper Steven , Greenwood Krista , Jackson-Humbles Daven , Bramble Lori , Harkema Jack , Wagner James , Tepe Jetze
Published: June 2021

Activation of nuclear factor‐ κB (NF‐ κB) has been linked to pulmonary inflammation and airway hyperresponsiveness. In previous studies we demonstrated that proteasome inhibition by a novel imidazoline (TCH‐013) inhibits NF‐ κB activation. To further characterize the role of the proteasome and NF‐ κB, we administered TCH‐013 during the effector phase of allergic airway disease. BALB/cJ mice were sensitized to ovalbumin (OVA) on Day 0, and boosted with OVA on Day 10. Mice were treated with TCH‐013 (i.p. 50mg/kg or vehicle) 1 hour prior to each OVA challenge on Days 17, 18 and 19. On Day 21, alterations in lung resistance and pulmonary inflammation were assessed. Treatment with TCH‐013 attenuated OVA‐induced increases in BALF total cellularity, eosinophils, and lymphocytes. Ovalbumin sensitized animals also exhibited increased IL‐5, IL‐6, IL‐13, KC, and airway resistance. These parameters were not affected by TCH‐013 administration. Proteasome inhibition by a TCH‐013 attenuated ovalbumin‐induced pulmonary inflammation but not airway hyperresponsiveness. We gratefully acknowledge support from Michigan State University and NIH (5R01CA142644).
PKCα‐dependent opening of the oxidant‐sensitive calcium channel TRPM2 induces apoptosis of lung endothelial cells
Authors: Hecquet Claudie M , Di Anke , Gao Xiaopei , Vogel Stephen M , Malik Asrar B
Published: June 2021

We addressed the relationship between oxidant‐activated transient receptor potential melastatin 2 (TRPM2) channel and its associated short splice variant (TRPM2‐S), in the gating of Ca2+, and how it mediates apoptosis of lung endothelial cells (LECs). We show that PKCα‐induced phosphorylation of TRPM2‐S prevents its association with TRPM2, and thereby activates Ca2+ gating activity. Results show that H2O2 induces association of PKCα with TRPM2‐S and phosphorylation of TRPM2‐S, and promotes dissociation of TRPM2‐S from TRPM2. Inhibition of PKCα activity and mutation of PKCα phosphorylation site (S39A) on TRPM2‐S N‐terminus prevents the uncoupling of TRPM2‐S from TRPM2‐L. Dissociation of TRPM2‐S from TRPM2‐L induces opening of TRPM2 channel and enables Ca2+ entry in LECs. This unique mechanism of TRPM2 channel activation was crucial for induction of oxidant mediated apoptosis of LECs. We conclude that oxidant‐induced LEC apoptosis is critically dependent of PKCα phosphorylation of TRPM2‐S and gating of Ca2+ via TRPM2; thus, pharmacologically maintaining TRPM2‐S and TRPM2 interaction by inhibiting PKCα activation, may be a novel means of preventing oxidative lung injury. Supported by AHA Grant SDG (10SDG2610057).
Kinins and Kinin Receptors in Pulmonary Vasoconstriction and Vascular Remodeling
Authors: Kuhr Frank K , Zimnicka Adrianna M. , Skidgel Randal A. , Christman John W. , Yuan Jason X.-J. , Levitan Irena
Published: June 2021

Kinins are vasoactive peptides that play important roles in vascular homeostasis, inflammation and angiogenesis. Although appreciated for their protective effects on the systemic circulation, kinins may be involved in the development of pulmonary hypertension (PH) as kinin antagonists have been shown to reduce pulmonary arterial pressure in hypoxic rats with severe pulmonary hypertension. Kinins activate two distinct G protein‐coupled receptors (GPCR), B2 receptors (B2R) which are expressed constitutively with a wide tissue distribution and B1 receptors (B1R) which are upregulated by inflammatory mediators, hypoxia or tissue injury. The endothelium‐dependent vasodilatative actions of kinins are well‐defined; however, the effect on pulmonary arterial smooth muscle cell (PASMC) contraction and proliferation is less understood. Our in vitro data show that B1R and B2R are both expressed in normal PASMCs and the mRNA expression of B1R is upregulated 2‐fold in PASMCs isolated from patients with idiopathic pulmonary arterial hypertension (iPAH). We have also found that kinins robustly activate the MAPK cascade and induce Ca2+ release in PASMCs. Taken together, our data suggest kinins may play a critical role in pulmonary vasoconstriction and vascular remodeling in iPAH and inhibition of kinin receptors may be an important target in developing novel therapies for PAH.

T32HL‐082547, HL‐066012
Effects of T‐ and B‐Cell Deficiency on Regulation of Hepatic P450 Enzymes in Mice Infected With an Intestinal Pathogen
Authors: Morgan Edward T. , Watkins William J. , Kinloch Ryan D , Nyagode Beatrice A.
Published: June 2021

Infections and inflammatory stimuli selectively regulate cytochrome P450 (P450) gene expression in the liver. While much attention has been devoted to the roles of the innate immune system and proinflammatory cytokines in this regulation, the influence of the adaptive immune system on the observed effects has not been addressed. Here, we address this question in severe combined immune deficiency (SCID) mice, which are deficient in T‐ and B‐cells. Female SCID or wild‐type mice in a C57BL/6 (B6) background were infected orally with C. rodentium, and hepatic P450 expression was studied 7 days later at the peak of infection. B6 and SCID mice were not different in colonic bacterial colonization or colonic mucosal hypertrophy, but SCID mice had 6‐fold higher levels of bacteria in their livers. Cyp4a10 and Cyp2b9 down‐regulation was attenuated in the SCID mice, while the regulation of other P450s was similar in the two strains. In mice with a C3H genetic background, the SCID mutation also blocked the down‐regulation of Cyp3a11 and Cyp2c29. These results indicate that bacterial translocation to the liver is not the cause of drug metabolizing enzyme down‐regulation, and suggest that B‐cells, T‐cells or their derived cytokines may have direct or indirect roles in the regulation of some hepatic P450 enzymes during infection. Supported by National Institutes of Health grant R01DK072372.
Changes in liver metabolic gene expression after radiation exposure
Authors: Wotring Virginia E , Peters Calvin P
Published: June 2021

Increased exposure to radiation is one physiological stressor associated with spaceflight. While known to alter normal physiological function, how radiation affects metabolism of administered medications is unclear. Crew health could be affected if the actions of medications used in spaceflight deviated from expectations formed during terrestrial medication use. Three different doses of gamma radiation (50 mGy – 6.05 Gy) and a sham were administered to groups of 6 mice each, and after various intervals of recovery time, liver gene expression was measured with RT‐qPCR arrays for drug metabolism and DNA repair enzymes. Results indicated ~65 genes of the 190 tested were significantly affected by at least one of the radiation doses. Many of the affected genes are involved in the metabolism of drugs with hydrophobic or steroid‐like structures, maintenance of redox homeostasis and repair of DNA damage. Most affected genes returned to near control expression levels by 7 days post‐treatment. With 6 Gy exposure, metallothionein expression was 132‐fold more than control at the 4 hr time point, and fell at each later time point (11‐fold at 24 hrs, and 8‐fold at 7 days). In contrast, Cyp17a1 showed a 4‐fold elevation at 4 hrs after exposure and remained constant for 7 days.
The Orphan Receptor/Corepressor DAX‐1 Modulates Human CAR Transcriptional Activity
Authors: Laurenzana Elizabeth M , Chen Tao , Sell Brian , Kannuswamy Malavika , Li Yong , Omiecinski Curtis J
Published: June 2021

The human constitutive androstane receptor (CAR) is a nuclear receptor (NR) primarily expressed in liver that regulates genes involved in xenobiotic metabolism, as well as lipid and energy homeostasis. Screening a panel of phage display peptides identified DAX‐1, a NR with corepressor properties, as a potential CAR coregulator. We next determined that DAX‐1 dose‐dependently inhibited CAR‐mediated transactivation. Directed mutation studies of DAX‐1 NR interaction domains showed that mutation of two N terminal domain ‘LXXM/LL’ motifs had no impact on DAX‐1 repression of CAR activity, whereas mutation of downstream ‘LXXLL’ or ‘PCFQVLP’ motifs restored CAR activity to 50% or 65% of control, respectively. Further, deletion of a transcription silencing domain (TSD) located in the DAX‐1 carboxy‐terminal region restored CAR transactivation to 90% of control levels. Two‐hybrid data demonstrated that full length DAX‐1 inhibited CAR‐SRC1 interaction by 50%, while the interaction was once again restored to 90% of control upon deletion of the TSD. Results of an alpha screen assay corroborated a direct interaction between CAR and DAX‐1 that was enhanced by CAR ligands. To examine ex vivo effects of DAX‐1, primary human hepatocytes were transfected with DAX‐1 and subsequent RT‐PCR analyses established that DAX‐1 inhibited CAR‐mediated induction of the CYP2B6 target gene by CITCO. In summary, these studies show that DAX‐1 is a functional corepressor of CAR transcriptional activity. Supported by NIH GM066411
Molecular mechanisms underlying netrin‐1 postconditioning induced cardioprotection against ischemia‐reperfusion injury
Authors: Bouhidel Jalaleddinne Omar , Cai Hua Linda
Published: June 2021

Given as a preconditioning mimetic, we have recently shown that netrin‐1 markedly reduces myocardial injury after I/R. Here, we aimed to investigate whether and how netrin‐1, exerts cardioprotection in vivo when given postconditionally. This is considered more clinically relevant. Eight‐weeks old DCC+/+ wild type mice and mice deficient in netrin‐1 receptor DCC (DCC+/−) were subjected to 30 min of myocardial ischemia and 10 min (analysis of signaling pathways and free radical production) or 24 h of reperfusion (analysis of infarct size). In DCC +/+ mice, treatment with 5 μg/kg netrin‐1 at the onset of reperfusion most significantly reduced infarct size, Ca2+‐induced mitochondrial swelling and superoxide production comparing to other dosages used (1, 10, 50 μg/kg), while it increased NO bioavailability and activation of cardioprotective proteins (ERK1/2, p70S6K, eNOS). All of these beneficial effects were abolished in DCC +/− mice, or under conditions of co‐treatment with UO126 (MEK inhibitor) or PTIO (NO scavenger). These seem to be consistent with our earlier ex vivo observations to examine a postconditioning effect of netrin‐1 (published abstract). Therefore the present study strongly suggests that netrin‐1 can function as a postconditioning mimetic to protect the heart against I/R injury, via a ERK1/2/eNOS pathway that also involves activation of DCC.
Resveratrol or 32°C hypothermia applied during reperfusion after cardiac ischemia reduces mitochondrial translocation of p66shc
Authors: Yang Meiying , Stowe David F. , Heisner James S. , Aldakkak Mohammed , Camara Amadou K.S.
Published: June 2021

Reactive oxygen species (ROS) are important in ischemia (I) and reperfusion (RP) injury. Mitochondria are sources of ROS via electron leak from complexes I and III. P66shc, a splice variant of ShcA adaptor protein family, enhances mROS by oxidizing reduced cyt c and reducing O2 to H2O2. Mild hypothermia or resveratrol (RES) reduce ROS during IRP. Although p66shc ablation protected against cardiac IR injury, it is unknown if and when p66shc is activated during IRP, and if inhibiting p66shc activation on RP protects against IRP injury. To test this, we isolated and perfused guinea pig hearts with KR buffer for 55 min (time control, TC), or 20 min or 35 min ischemia, or 35 min ischemia plus 20 min RP, with or without hypothermia or RES (10 μM). We isolated heart tissue total cell lysates, mitochondria, and cytosol and measured levels of p66shc by Western blot (WB) with anti‐SHC antibody and phosphorylation of Serine36 of p66shc by immunoprecipitation with anti‐SHC followed by WB with p66‐ Ser36 antibody. We found that after RP mitochondrial p66shc increased two fold above TC while phosphorylation of Ser36 increased at 20 min ischemia, but decreased at 35 min ischemia, and increased again at 20 min RP in total cell lysates. Our results demonstrate that p66shc is activated by IRP, that activated p66shc translocates from cytosol to mitochondria, and that RES or hypothermia applied only during RP reduces mitochondrial p66shc to TC levels.
Fasting Glucose Level Differences Modulate Cell Surface Expression of CD11b and CD66b in Granulocytes and Monocytes in Type II Diabetic Subjects
Authors: HORVATH PETER , OLIVER STACY R. , GANESAN GOUTHAM , ZALDIVAR FRANK , RADOMAIZIK SHLOMIT , GALASSETTI PIETRO R.
Published: June 2021

Cardiovascular complications are the leading cause of mortality in type II diabetes (T2D), in which onset and progression of atherosclerosis has been associated with chronic inflammation, now believed to be at least in part modulated by the activation status of innate immune cells such as granulocytes (Gc) and monocytes (Mc). Activation of these cells (as reflected for instance by increased CD11b and CD66b surface markers) increases their adhesion ability to the endothelium and their release of other cytokines/chemokines. While inflammatory activation has been associated with poor glycemic control, the effect of acute spontaneous hyperglycemia on innate immune cell activation remains unclear. To address this issue, whole blood of healthy and T2D male subjects with low and high fasting blood sugar was incubated for 0, 30, 90 and 240 min, and expression of key markers (CD11b, CD14, CD16, CD62L, CD66b) was determined by flow cytometry. In the T2D group, subjects with high fasting glycemia had significantly higher Gc CD11b and CD66b surface mean fluorescence intensity (MFI) as compared to T2D subjects with normal glycemia, whose values were identical to healthy controls (see figure). Our data suggests that, independent of the presence of diabetes per se, acute hyperglycemia seems to be a major contributor to innate cell inflammatory status, and their consequent endothelial adhesion and vascular damage potential.

Supported by NIH Grants P01HD048721 and UL1RR031985
Leukocyte Mechanosensitivity to Fluid Shear Stress Depends on the Subtypes of CD18 Integrins
Authors: Zhang Xiaoyan , Zhan Dongying , Shin Hainsworth Y.
Published: June 2021

Fluid shear stress cleaves CD18 integrins on neutrophils, hypothetically to avert their adhesion to other cells such as platelets. Since lymphocytes and monocytes also express CD18 integrins (mainly LFA‐1 and Mac‐1) but in a cell type specific fashion, we examined whether shear‐induced CD18 cleavage is different among the three leukocyte subsets and if this shear response prevents leukocyte‐platelet adhesion. We exposed mixed leukocyte populations to shear stress (2.5 – 10 dyn/cm2; 1 – 10 min) to assess cell and receptor subtype‐specific CD18 cleavage. Shear stress cleaved Mac‐1 on neutrophils and monocytes, but LFA‐1 on lymphocytes devoid of surface Mac‐1. Furthermore, neutrophils exposed to shear after treatment with cysteine protease inhibitor E‐64 (which blocks CD18 cleavage) exhibited elevated platelet adhesion and an impaired CD18 cleavage response relative to naïve cells. But, neither monocytes nor lymphocytes exhibited increases in platelet adhesion or impaired CD18 cleavage after E64 treatment. Shear‐induced CD18 cleavage and its impact on platelet adhesion thus appear to be cell type‐specific contingent on their differential expression of CD18 integrins and proteases. In terms of neutrophils, our data further support fluid shear mechanotransduction as an anti‐inflammatory measure under physiologic conditions. Funded by the NSF‐KY EPSCoR, and AHA.
Chronic Nicotine (NIC) Aggravates Sub Pressor Angiotensin II (SP‐AngII)‐Induced Renal Hemodynamics And Resistance Vessel Remodeling
Authors: Maranon Rodrigo Oscar , Chandrashekar Kiran , Lopez-Ruiz Arnaldo , Soljancic Andrea , Arany Istvan , Liu Ruisheng , Juncos Luis A
Published: June 2021

Smoking can worsen SP‐AngII induced chronic kidney disease (CKD). However, the mechanism behind this is equivocal. We hypothesized that NIC remodels the renal resistance vessels, worsening the hemodynamic parameters and hence contributes to the eventually deterioration of CKD. Male SD rats were pretreated for 12 days with NIC (12.5μg/ml) or vehicle (saccharine 2%) in water and then randomized into 4 groups: 1) Control (Cont), 2) NIC, 3) SP‐AngII (200ng/kg/min), 4) SP‐AngII + NIC. Each group received either vehicle or SP‐AngII by SQ osmotic minipumps, for an additional 22 days. Renal hemodynamics were measured in anesthetized animals, the kidneys harvested for morphometric analysis of afferent arteriole (Af‐Art) remodeling.

Groups
Proteinuria (mg/24 hrs)
Medial Layer of Af‐Art (μm2)
Lumen of Af‐Art (μm2)
Media/Lumen ratio
RVR (ml/min/mmHg)
CRVR (TPU/mmHg)
MVR (TPU/mmHg)

Cont
19±1.2
124.3±12
144.5±6.3
1.13±0.1
16.2±2.5
2.6±0.2
4.5±0.2

NIC
34±4.4*
130.0±11
156.7±6
0.82±0.1
15.7±0.5
2.5±0.19
4.9±0.2

SP‐AngII
99±8.9†
253.1±11†
75.69±2.4†
3.55±0.2†
23±1.6†
3±0.17
6.6±0.2†

SPAngII+NIC
150±6.5#†
321.3±10#†
31.53±2#†
11.98±1#†
32±2#†
3.6±0.2#†
9.5±0.4#†

p< 0.05: * vs. Veh; # vs. SP‐AngII; † vs. NIC

NIC worsened the SP‐AngII‐induced renal damage, and exacerbates the vascular resistance vessel remodeling that may contribute to the worsening hemodynamic parameters and eventually deterioration of Sp‐AngII‐ induced CKD.
Vascular adaptation and network efficiency
Authors: Tyrrell James Alex , Kunert Christian , Gruionu Gabriel , Munn Lance L
Published: June 2021

The formation of stable vascular networks is a complex process, and the exact mechanisms involved are not known. In addition to shear‐driven adaptation, tissue‐derived growth factors are thought to be important for maintaining a stable vasculature. Thus, the minimization of the energy necessary to drive flow can be balanced against the metabolic demands of the surrounding tissue. Using the principle of parsimony, we seek the simplest mathematical model of structural adaption that leads to a stable, physiologically plausible equilibrium. To simulate vascular remodeling, we employ a lattice Boltzmann framework (LBM) optimized for graphics processing units (GPUs) and incorporate numerical techniques derived from image processing to track the evolution of the computational domain over time. Some novel aspects of our model include: i) spatially‐resolved mesoscale permeabilities for both fluid and solute species; ii) particle erythrocyte advection with real oxygen release dynamics; iii) diffusive and conductive signaling; iv) automatic handling of changes in vascular topology. In addition to simulating structural adaption in silico, we combine our modeling framework with in vivo imaging to analyze the structure and function of vessels in living tissues.
Bone marrow cells are necessary for cerebral microvascular recovery following whole brain radiation therapy in mice
Authors: Warrington Junie Paula , Csiszar Anna , Mitschelen Matthew , Yan Han , Han Song , Sosnowska Danuta , Gautam Tripti , Farley Julie A , Lee Yong Woo , Ungvari Zoltan , Sonntag William E
Published: June 2021

Whole brain radiation therapy (WBRT), used to treat metastatic brain tumors, induces brain microvascular rarefaction and is important in the genesis of cognitive impairment in survivors. The etiology for the absence of microvascular recovery post‐WBRT is not well established.

We hypothesized that WBRT inhibits, while hypoxia stimulates, hematopoietic progenitor cell (HPC) mobilization from bone marrow (BM) and once mobilized, cells home to brain and associate with blood vessels.

C57BL/6 mice were subjected to 1 or 8 fractions of 4.5Gy WBRT, twice a week for 4 weeks. We found that a single dose of WBRT increased BM HPCs (CD45+/CD34+/CD133+/VEGFR2+) but release into the circulation was impaired. However, following systemic hypoxia (11% oxygen for 7 days), HPCs were mobilized. To determine whether circulating BMDCs home to brain and to establish their phenotype, C57BL/6 mice received BM transplant from C57BL/6‐Tg(UBC‐GFP)30Scha/J mice. BMDCs were evident in the hippocampus, developed a microglial phenotype, and associated with BrdU+ microvessels. Importantly, BMDCs did not develop an endothelial phenotype.

Our results are consistent with the hypothesis that lack of cerebral microvascular recovery post‐WBRT results from impaired release of BM HPCs. Systemic hypoxia repopulates brain peri‐vascular microglia and re‐establishes a microenvironment conducive to angiogenesis.
Obesity Induced Collateral Growth Impairment: Potential Role of Endothelial Activation and Leukocyte Mobilization/Recruitment
Authors: Distasi Matthew , Campana Gonzalo , Ortiz Miguel , Labarrere Carlos , Miller Steven , Ingram David , Unthank Joseph
Published: June 2021

To assess potential mechanisms responsible for the impairment of collateral growth observed in obesity, we evaluated endothelial activation and leukocyte mobilization and recruitment in lean and obese mice after focal femoral artery occlusion. Immunoreactivity for ICAM‐1 and F4/80, markers of endothelial activation/inflammation and macrophages respectively, was significantly increased in collateral gracilis arteries at 3 days post‐ ligation (P<0.001) and was greater in obese than lean mice (P<0.01). FACS analysis of circulating cells at baseline (Day 0) and 3 days post‐ligation is presented in the table and indicates major differences between specific cell populations that are mobilized and/or recruited after femoral artery ligation in lean and obese mice. In conclusion, impaired collateral growth in obese mice is associated with enhanced, rather than suppressed endothelial activation and macrophage recruitment, and significant differences exist in the specific bone marrow derived cells that are mobilized and recruited.

Grant Funding Source : NIH T32 HL007910‐26
Vasopressin levels in patients undergoing pulmonary thromboendarterectomy (PTE)
Authors: Nguyen Liem C , Banks Dalia , Manecke Gerard , Shurter Jesse , Schilling Jan , Patel Hemal , Roth David R
Published: June 2021

Vasopressin is a major determinant of vascular tone during cardiac surgery however vasopressin levels are unknown in patients presenting for pulmonary thromboendarterectomy (PTE) utilizing cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA). We performed an observational study in 22 patients with chronic thromboembolic disease undergoing PTE with CPB and DHCA to assess perioperative vasopressin levels. Serum vasopressin levels were assessed using a radioimmunoassay at: 1. baseline before surgery, 2. after anesthetic induction, 3. during CPB, 4. after DHCA, 5. after CPB. In 8 patients, vasopressin was undetectable (< 0.5 pg/mL) at baseline. Vasopressin before and after induction of anesthesia was 1.06 +/− 0.96 (SD) pg/mL and 1.21 +/− 1.54 pg/mL, respectively. During CPB, vasopressin increased to 19.22 +/− 28.22 pg/mL (a 2–30‐fold increase from baseline). Vasopressin remained elevated after DHCA (16.29 +/− 22.71 pg/mL) and after CPB (23.619 +/− 26.29 pg/mL). There were no differences between vasopressin levels during CPB, after DHCA, or after CPB. We conclude that a significant portion of these patients may present for PTE with a relative deficiency of vasopressin. Nevertheless, there is a marked increase from baseline in the secretion of vasopressin at the initiation of CPB and thereafter. Further studies are required to determine if vasopressin secretion in this population is abnormal.
Sympathetic neural and hemodynamic responses to upright tilt in elderly African Americans versus Caucasians
Authors: Fu Qi , Okada Yoshiyuki , Galbreath M. Melyn , Jarvis Sara S. , Bivens Tiffany B. , Meier Rhonda L. , Vongpatanasin Wanpen
Published: June 2021

It has been demonstrated that neural control of blood pressure (BP) is different in young African Americans (AA) and Caucasians (CAU). Whether this is also true in elderly people is unclear. We tested the hypothesis that elderly AA have enhanced sympathetic neural responses during orthostasis compared with elderly CAU. Muscle sympathetic nerve activity (MSNA), BP, and heart rate (HR) were recorded continuously, cardiac output (Qc, C2H2 rebreathing), stroke volume (SV=Qc/HR) and total peripheral resistance (TPR=mean BP/Qc) were measured intermittently supine and during 5‐min 60‐degree upright tilt in 10 AA [6 M, 4F; 65±4 (SD) yrs] and 15 CAU (9M, 6 F; 67±4 yrs). AA and CAU had similar supine (44±10 vs 42±8 bursts/min) and upright MSNA burst frequency (59±11 vs 60±11 bursts/min; P=0.98 for race, <0.01 for posture, 0.58 for interaction). However, upright total MSNA was smaller in AA than CAU (162±39 vs 248±126%; P<0.01). Systolic BP, HR, Qc and SV were not different between groups in the supine position and during upright tilt. Supine diastolic BP was similar between groups (66±7 vs 65±8 mmHg; P=0.76), but upright diastolic BP was greater in AA than CAU (80±10 vs 71±8 mmHg; P=0.02). TPR did not differ between AA and CAU in both supine (1536±295 vs 1741±401 dyn·s·cm−5) and upright postures (2490±555 vs 2522±886 dyn·s·cm−5; P=0.47 for race, <0.01 for posture, 0.71 for interaction). These results suggest that elderly African Americans have a blunted sympathetic neural responsiveness but enhanced blood pressure reactivity during orthostasis compared with elderly Caucasians, which may be attributable to an augmented sympathetic vascular transduction.

Supported by NIH R01 HL091078.
Influence of 24‐hour sleep deprivation on anxiety and cardiovascular reactivity in humans
Authors: Larson Robert A , Durocher John J , Yang Huan , DellaValla Joseph P , Carter Jason R
Published: June 2021

Hypertensive responses to 24‐hour total sleep deprivation (TSD), self‐perceived anxiety, and mental stress have been studied independently, but not collectively. We hypothesized that TSD‐induced anxiety would be associated with increased resting mean arterial pressure (MAP) and cardiovascular reactivity to acute mental stress (5 min of mental arithmetic). State‐anxiety, MAP, heart rate (HR), and forearm vascular conductance (FVC) were measured in 28 subjects (14 men, 14 women; age 22±1 yrs) after normal sleep (NS; 7±0 hrs) and after 24‐hour TSD (randomized, crossover design). TSD increased state‐anxiety (28±1 to 33±1 units; P<0.01) and resting MAP (72±1 to 75±1 mmHg, P<0.01), but increases in MAP were not correlated with increases in state‐anxiety (r=0.17, p=0.20). Mental stress elicited similar increases (P<0.01) in MAP (Δ9±1 vs. Δ9±1 mmHg), HR (Δ15±1 vs. Δ18±2 beats/min), and FVC (Δ1.2± 0.2 vs. Δ1.2±0.2 units) during NS and TSD, respectively. Increases in state‐anxiety were not associated with cardiovascular reactivity to mental stress. When probed for sex differences, men and women demonstrated similar response patterns. In conclusion, TSD elicits concurrent increases in state‐anxiety and resting MAP, but these responses appear to occur independently. Moreover, TSD‐induced state‐anxiety does not alter cardiovascular reactivity to mental stress. Supported by NIH (HL‐ 098676).
A novel telemeter for chronic measurement of arterial blood pressure or ECG in mice
Authors: Lim Matthew , Russell David , Pauly Bob , McCormick Daniel , Kirton Robert , Kondo Masahiro , Guild Sarah-Jane , Malpas Simon , Budgett David
Published: June 2021

Currently telemetry devices for recording of physiological signals in mice are restricted by the relatively short battery life necessitating expensive exchange. We have developed a fully implantable telemetry system for high fidelity measurement of arterial pressure or ECG in mice. For recording arterial pressure the system incorporates a 1Fr micro‐tip solid state pressure catheter (diameter 0.3 mm) developed specifically for this device. The telemeter is continuously powered by a magnetic field, eliminating the requirement for a battery and thus allowing for potentially lifetime implantation and telemeter reuse. Data is sampled at 2 kHz enabling high fidelity recordings of arterial or ECG waveforms. Such high frequency sampling is particularly relevant for the analysis of ECG parameters. The system function was validated by implantation into mice, 25 gm or larger, and recording data for up to one month. The system developed is suitable for making arterial pressure measurements from mice, and could expand the scope of chronic monitoring.
PLCγ1 is Required for IP3‐mediated Activation of TRPM4 in Cerebral Artery Smooth Muscle Cells
Authors: Gonzales Albert L , Sanders Lindsey , McPhaul Alainna J , Earley Scott
Published: June 2021

In cerebral artery myocytes, the melastatin transient receptor potential channel TRPM4 is activated by Ca2+ release from proximal inositol trisphosphate receptors (IP3R), and is essential for pressure‐induced membrane depolarization and vasoconstriction. Multiple isoforms of three families of phospholipase C (PLC) isoforms are present in vascular smooth muscle cells (β, γ, and δ). All of these enzymes cleave phosphotidylinositol (4,5)‐bisphosphate (PIP2) to generate the second messengers diacylglycerol (DAG) and inositol 1,4,5‐ trisphosphate (IP3). We tested the hypothesis that PLC is required for IP3R‐mediated activation of TRPM4 channel activity. We observe a dose‐dependent increase in TRPM4 channel activity in the presence of membrane permeable IP3 analog Bt‐IP3. TRPM4 channel activity is inhibited by the general PLC blocker U‐73122 and rescued following administration of Bt‐IP3. The IP3R inhibitor Xestospongin C inhibited Bt‐IP3‐dependent activation of TRPM4. To characterize the specific PLC isoform responsible, we used RNAi technology to knockdown PLCγ1 expression in cerebral arteries, and observe a loss of TRPM4 channel activity in myocytes isolated from PLCγ1 siRNA‐treated arteries. These findings suggest that PLCγ1 is responsible for generation of IP3 and activation of TRPM4 in cerebral artery smooth muscle cells. RO1HL091905(SE); F31HL094145‐01 (AG).
Dihydrotestosterone suppresses oxidative stress and improves vascular function following induction of inflammation in the cerebral circulation
Authors: Gonzales Rayna , Techapinyawat Rheana , Zuloaga Kristen
Published: June 2021

During pathophysiological events (i.e. stroke) enzymatic actions of cyclooxygenase‐2 (COX‐2) and inducible nitric oxide synthase result in reactive oxygen species (ROS) generation that could potentially alter vascular function. Since dihydrotestosterone (DHT) augments COX‐2 in the absence of inflammation and attenuates COX‐2 in the presence of inflammation in rat and human vascular smooth muscle (VSM), we hypothesized that DHT would attenuate ROS and thus improve vascular function following an inflammatory insult. Oxidative stress was assessed in primary human brain VSM cells stimulated with cytokine in the absence or presence of DHT (18 h; 10 nM) using ROS‐indicator dyes (H2DCFDA and DAF‐FM). Myogenic tone was measured in middle cerebral arteries (MCA) isolated from castrated male rats treated with DHT (2 wk; 25 mg pellet/21 days) followed by endotoxin (6 h; 0.5 mg/ml i.p.). In VSM cells, DHT attenuated cytokine‐induced ROS production. In MCA, chronic DHT treatment increased pressure‐induced tone. However, in the presence of endotoxin, DHT's ability to increase tone was blunted without altering the myogenic response to step pressure increases. These data suggest that DHT may provide protection against inflammation‐induced injury in the cerebrovasculature in part by decreasing ROS (e.g. peroxynitrite and hydrogen peroxide) and thus improving vascular function. Sarver Heart Center & AHA (RG & KO)
Antioxidant roles of heme oxygenase, carbon monoxide and bilirubin in cerebral circulation during epileptic seizures
Authors: Parfenova Helena , Leffler Charles W. , Basuroy Shyamali , Fedinec Alex L.
Published: June 2021

Epileptic seizures cause sustained loss of endothelium‐dependent regulation of cerebral blood flow. Heme oxygenase (HO) is cytoprotective against seizure‐induced cerebral endothelial dysfunction. Oxidative stress is a potential cause of vascular damage. Using a model of bicuculline‐induced epileptic seizures in newborn pigs, we addressed the hypotheses that: 1) seizures increase production of reactive oxygen species (ROS) in cerebral vasculature, 2) endogenous HO activity exerts antioxidant effects, and 3) CO and bilirubin exhibit antioxidant properties in cerebral circulation. As assessed by dihydroethidium (DHE) oxydation, seizures elevate ROS in cerebral vessels. Burst of ROS during seizures was blocked by tiron, a ROS scavenger, and reduced by apocynin, a NADPH oxidase inhibitor. A HO inhibitor, tin protoporphyrin, potentiated whereas a HO‐1 inducer, cobalt protoporphyrin, blocked the ROS increases in cerebral vessels during seizures. The products of HO activity, CO (as CORM‐A1, 2 mg/kg ip) and bilirubin (3–5 mg/kg, iv) administered before or during a 20‐min window after seizure onset attenuated ROS elevation. These data indicate that: 1) constitutive HO‐2 and inducible HO‐1 have antioxidant properties in cerebral vasculature; 2) antioxidant properties of CO and bilirubin could contribute to the HO‐mediated endothelial survival mechanism in cerebral circulation during epileptic seizures.
White matter hyperintensities are associated with surrogate markers of cerebrovascular risk
Authors: Raz Limor , Kantarci Kejal , Miller Virginia M
Published: June 2021

White matter hyperintensities (WMHs) are associated with cerebral microvascular disease. Our objective was to investigate changes in volume of WMH in relationship to conventional cerebrovascular risk factors in recently menopausal women (within 6 months – 3 yrs of their last menses) enrolled in the Kronos Early Estrogen Prevention Study (KEEPS). Platelet activation, numbers of platelet‐derived microvesicles and conventional risk factors for cerebrovascular disease were determined in women (105) enrolled in KEEPS at Mayo Clinic. Each woman underwent MRI of the brain and WMH volumes were derived from the segmented Fluid Attenuated Inversion Recovery images at baseline and 36 months after randomization to treatment. Parametric, nonparametric and log transformation statistical analyses were conducted appropriately. Blood pressure and lipid profiles at baseline were not associated with changes in WMHs. However, baseline platelet micro‐aggregates, annexin V positive microvesicles and plasma activated protein C significantly correlated with increases in WMHs at 36 months. In conclusion, this correlation of increase in WMH with markers of platelet activation measured at baseline suggests that activated platelets and factors contributing to platelet activation may be causal to development of WMH. Supported by the Aurora Fnd, NIH HL90639, NS066147, HD65987 and the Mayo Fnd.
Defining cerebral autoregulation: is dynamic simply faster static?
Authors: Tan Can Ozan
Published: June 2021

The cerebrovasculature uniquely autoregulates flow in the face of changing pressure. The static curve established that cerebral autoregulation is nonlinear, encompassing a region where pressure changes are counterregulated, and outside of which pressure induces parallel flow changes. Recent work has shown low coherence (i.e., frequency specific linear correlation) between pressure and flow fluctuations, indicating a ‘dynamic’ autoregulation. However, low coherence also evidences insufficiency in linear analyses ‐ absence of correlation prohibits assessment of autoregulatory function. Therefore, I applied a nonparametric projection pursuit regression to extract the dominant nonlinearity within cerebral flow response to pressure fluctuations. Data was obtained from 48 volunteers during oscillatory lower body negative pressure (30–40 mmHg) at 6 frequencies (0.03–0.08 Hz). The regression explained the majority of flow changes at all frequencies and provided a nonlinearity that was consistent across all individuals and that diminished at higher frequencies (Figure). This characteristic nonlinearity accounts for low coherence and suggests that dynamic autoregulation simply may reflect a ‘faster’ static function. Thus, a robust yet simple nonlinear approach can enhance our integrative understanding of cerebral autoregulatory control. Supported by NIH grant HL093113.