bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of phosphodiesterase‐4 (PDE4) in alcohol‐ and heroinseeking behavior
Authors: Zhang Han‐Ting , Lai Miao‐Jun , Wen Rui‐Ting , Gong Mei‐Fang , Hu Wei , Zhou Wen‐Hua
Published: June 2021

Cyclic AMP (cAMP) signaling is important in mediating drug abuse and dependence, including alcoholism. As a critical controller of intracellular cAMP levels in the brain, phosphodiesterase‐4 (PDE4), an enzyme that specifically hydrolyzes cAMP, may play a role in drug dependence. Here we examined the effects of rolipram, a selective PDE4 inhibitor, on alcohol or heroin seeking behavior in mice and/or rats. Treatment with rolipram (0.1–0.5 mg/kg) decreased ethanol (7–12%) intake and preference in high alcohol preferring mice and rats in two‐bottle choice and/or ethanol drink‐in‐dark tests. Deficiency of PDE4B diminished rolipram‐induced decreases in ethanol consumption in mice. In addition, rolipram (0.025–0.05) also decreased ethanol responses in operant self‐administration in rats. Further, in heroin self‐administration, rolipram (0.03–0.3 mg/kg) decreased cues‐ or heroin priming‐induced responses, but did not alter responses to heroin injections. These results suggest that PDE4 plays an important role in the regulation of ethanol‐ and heroin‐seeking behaviors, which are attenuated by PDE4 inhibitors such as rolipram. Targeting PDE4, in particular PDE4B, may be an efficient approach for treatment alcoholism and heroin dependence [This work was supported by research grants from NIH/NIAAA (AA020042; to HTZ), National Nature Science Foundation of China (30870894; to JHL/81071077; to WHZ), and National Basic Research Program of China (2009CB522008; to WHZ)].
Cross‐species and Painless: Targeting truncated mu opioid receptor splice variants in the rat
Authors: Grinnell Steven , Majumdar Susruta , Pasternak Gavril W
Published: June 2021

We recently synthesized a highly potent analgesic, 3′‐Iodobenzoylnaltrexamide (IBNtxA), which exhibited a favorable side effect profile devoid of respiratory depression, dependence, or rewarding behavior in a conditioned place preference assay. Analgesia was retained in a triple knockout mouse lacking traditional mu, delta, and kappa opioid receptors but retaining exon 11‐associated splice variants of MOR‐1; this analgesia was lost in an MOR‐1 exon‐11 knockout animal, implicating these variants in the drug's actions. We sought to determine whether IBNtxA exhibits similar pharmacology in the rat. IBNtxA retained high analgesic potency, and did not show any signs of respiratory depression. Saturation and competition studies in rat brain using 125IBNtxA demonstrated a site possessing a similar pharmacological profile to the site observed in mouse brain. We conclude that this target is not unique to the mouse, and that given the highly conserved splicing between the rat and human MOR‐1 genes, it is likely to have significant clinical relevance. This work was supported by a fellowship from the PhRMA Foundation to SG and grants from the National Institute on Drug Abuse (DA06241, DA02165, and DA07242) to GP.
TY032, a potent opioid agonist/neurokinin 1 antagonist produces analgesia without motor impairment or sedation
Authors: Sandweiss Alex , Faridian Martin Kosar , Hu Jackie , Wallace Scott , Ortega Saul , Davis Peg , Ondoua‐Lozano Alysia , Lee Yeon Sun , Hruby Victor J , Vanderah Todd W
Published: June 2021

The use of opioids for the treatment of pain has been restricted by their side effects including constipation, somnolence, antinociceptive tolerance, addiction, and respiratory depression. Here, we use an in vivo model of neuropathic pain to characterize TY032, a bifunctional pharmacophore containing both opioid agonist and Neurokinin 1(NK1) antagonist activity. Following spinal nerve ligation (SNL) to induce neuropathic pain in rats, intrathecal (i.t.) administration of TY032 produced statistically significant dose dependent antiallodynia to innocuous mechanical stimulation with a peak effect 80 min post‐injection. Similarly, dose dependent antihyperalgesia and analgesia to thermal stimuli were achieved in rats following i.t. administration of TY032, with the peak effect 20 min after injection. Naloxone attenuated the analgesic effect of TY032 in a tail‐flick model of thermal pain. With naloxone on board, i.t. administration of TY032 in mice significantly reduced the average number of flinches during a 5 min period immediately following Substance P administration. Finally, TY032 had no effect on motor impairment or sedation using rotarod. Studies here put forth the idea that a non‐addictive, non‐constipating opioid can and should be made available for both patients suffering from chronic pain and physicians’ uneasiness in prescribing narcotics.
The effect of glutamine and alanylglutamine as substrates for the isolated perfused rabbit kidney
Authors: Fonteles Manasses Claudino , Sousa‐Filho Jose Ernando , Nascimento Nilberto Robson Falcão , Araujo‐Filho Raimundo Messias
Published: June 2021

Rabbit kidneys were perfused with a high K+ and Mg++ solution as previously described (J S R 21(3);169,1976). Briefly the animals were anesthetized with urethane and pentobarbital and the left kidney perfused at 37ºC with a previously dialyzed solution in an artificial heart‐lung machine custom fabricated. Several functional physiological parameters were evaluated such as perfusate and urine flow, GFR, %TNa, %TK, osmolar clearance, perfusion pressure and vascular resistance. The experiments lasted 2 hours. To measure kidney function creatinine was added to the perfusate and Na,K, and osmolarities were measured every ten minutes. Urinary flow increased 6.5‐fold in AG group when compared to control. Glutamine increased this parameter 16.5 times. The same trend was observed with GFR that was enhanced 5.5 times in the AG group while Glut 1 increased 10.6%. The fractional sodium reabsorption increased 8.87% with AG while Glut increased 11.26%. No changes were observed in the fractional transport of potassium. We conclude that these 2 substrates promote a great improvement in kidney functional preservation in this ex vivo model. These substrates could be important metabolic support when perfusing kidney for transplantation.

Financial support: CNPq and FUNCAP Brazil
Mitochondrial‐targeted antioxidants represent a promising approach for prevention of cisplatin‐induced nephropathy
Authors: PACHER PAL , Mukhopadhyay Partha , Horvath Bela , Zsengeller Zsuzsanna , Zielonka Jacek , Kechrid Malek , Stillman Isaac E , Parikh Samir M. , Joseph Joy , Kalyanaraman Balaraman
Published: June 2021

Cisplatin is a widely used antineoplastic agent; however, its major limitation is the development of dose‐dependent nephrotoxicity whose precise mechanisms are poorly understood. Here we show not only that mitochondrial dysfunction is a feature of cisplatin nephrotoxicity, but also that targeted delivery of superoxide dismutase mimetics to mitochondria largely prevents the renal effects of cisplatin. Cisplatin induced renal oxidative stress, deterioration of mitochondrial structure and function, an intense inflammatory response, histopathological injury, and renal dysfunction. A single systemic dose of mitochondrially targeted antioxidants, MitoQ or Mito‐CP, dose‐dependently prevented cisplatin‐induced renal dysfunction. Mito‐CP also prevented mitochondrial injury and dysfunction, renal inflammation, and tubular injury and apoptosis. Despite being broadly renoprotective against cisplatin, Mito‐CP did not diminish cisplatin's antineoplastic effect in a human bladder cancer cell line. Our results highlight the central role of mitochondrially generated oxidants in the pathogenesis of cisplatin nephrotoxicity. Because similar compounds seem to be safe in humans, mitochondrially targeted antioxidants may represent a novel therapeutic approach against cisplatin nephrotoxicity.
Allopathic and Ayurvedic approaches in management of vitiligo
Authors: Senthilkumar Uma , Mohanram Arun
Published: June 2021

Vitiligo an acquired disorder of pigmentation affects up to 2% of the world's population. The etiopathogenesis of this disease is better understood now than a decade ago. This abstract aims at highlighting the role of both modern and complementary medicine in the management of vitiligo from the literature. Recent advances on the modern medical front are Narrowband Ultraviolet B (NBUVB, Targeted Ultraviolet B (UVB), Excimer laser therapies, topical calcineurin inhibitors, topical pseudocatalase, and topical Vitamin D analogues in combination with Ultraviolet (UV) light. The surgical options include suction and split grafting and melanocyte culture transplantation. On the complementary front, many trials have investigated the use of Ayurvedic plants in the treatment of vitiligo. These trials studied photosensitizing agents from Picorrhiza kurroa, a khellin extract, and Polypodium leucotomos. Psoralea corylifolia extract is known to stimulate to melanoblasts and is commonly used in the management of vitiligo. The use of oral Ginkgo biloba as monotherapy for vitiligo is also promising. Ginkgo's efficacy without the need for phototherapy thereby eliminating the adverse effects inherent with phototherapy makes it a therapeutic option worth investigating. Still further investigations and more controlled trials studying effect of plant extracts in the treatment of vitiligo are needed.
Reduction in central mitochondrial ROS improves cardiovagal baroreflex function independent of blood pressure in (mRen2)27 rats
Authors: Nautiyal Manisha , Shaltout Hossam A , Chappell Mark C , Diz Debra I
Published: June 2021

Mitochondria‐derived reactive oxygen species (ROS) are potential therapeutic targets in hypertension. Transgenic (mRen2)27 rats overexpressing the murine Ren2 gene are hypertensive with impaired baroreflex sensitivity (BRS) for control of heart rate (HR). These animals also show elevated mitochondrial ROS in brain dorsal medulla, an important site for modulation of BRS. To determine whether central mitochondrial ROS influences mean arterial pressure (MAP) and BRS, the mitochondria‐targeted ROS scavenger Mito‐TEMPO (MT; 0.15 mg/day for 4 weeks) or artificial cerebrospinal fluid (aCSF) were infused ICV in 18 week old male (mRen2)27 rats. MT treatment failed to lower MAP but reduced HR [400 ± 23 vs. aCSF: 450 ± 7 bpm; p = 0.03; n = 4–7]. Lowering of HR was associated with increased vagal components of the spontaneous BRS [Sequence UP: 0.2 ± 0.2 vs. aCSF: −0.2 ± 0.1 ms/mm Hg; p = 0.03] and HR variability [SDRR: 7.6 ± 2 vs. aCSF: 3.3 ± 0.6 ms; p = 0.02]. Overall sympathovagal balance was reduced [LFRRI/HFRRI: −0.1 ± 0.1 vs. aCSF: 0.3 ± 0.1, p = 0.03]. Central scavenging of mitochondrial ROS improved BRS independent of MAP alterations in the (mRen2)27 rats. Since impaired BRS significantly associates with cardiovascular pathologies, both targeting of mitochondrial ROS and lowering of MAP may provide optimal therapeutic benefit.
Sulfuraphane (SFN) activation of nuclear factor E2‐related factor 2 (Nrf2) increases expression and transport activity of P‐glycoprotein (P‐gp) and breast cancer related protein (Bcrp) at the blood‐brain barrier (BBB)
Authors: Miller David S. , Cannon Ronald E , Peart John , Campos Christopher R , Smith Lindsay K , Wang Xueqian
Published: June 2021

Nrf2 is a redox‐sensitive transcription factor that plays a critical role in cellular defenses against oxidative/electrophilic stress. Administering SFN, a Nrf2 ligand present in cruciferous vegetables, is neuroprotective in cerebral ischemia, traumatic brain injury and subarachnoid hemorrhage; Nrf2 is proposed as a therapeutic target. Here we assess the effects of Nrf2 activation on the transport activity and expression of the BBB drug efflux pumps, P‐gp and Bcrp using isolated rat and mouse brain capillaries. Capillaries from rats dosed with SFN (10 mg/kg i.p.) exhibited 2–3‐fold increased P‐gp transport activity and P‐gp and Bcrp protein expression. Exposing isolated brain capillaries to 0.1–1 uM SFN for 3 h increased P‐gp and Bcrp activity and expression. Inhibiting transcription or translation abolished upregulation of transport. In brain capillaries from Nrf2‐null mice, SFN did not alter transport activity. Surprisingly, Nrf2 signaled indirectly through p53 and NF‐kB to increase transporter activity/expression. Thus, a consequence of Nrf2‐induced neuroprotection is selective tightening of the BBB to therapeutic drugs. Supported by the Intramural Research Program NIH/NIEHS.
HUMAN HEPATOCYTE INDUCTION STUDIES TO EVALUATE POTENTIAL OF CYP3A4 INDUCTION IN VIVO: TWENTY COMPOUNDS WITH THREE DONORS
Authors: Stresser David M , Ho Thuy , Callendrello Alanna , Clark Robert J , Santone Elizabeth , Fox Lisa G , Zhang George
Published: June 2021

Recent guidance from regulatory agencies advocates the use of human hepatocytes as the test system for evaluating cytochrome P450 induction potential of drug candidates. The use of hepatocytes which have been prequalified for response to known clinical inducers and non‐inducers are indicated. In addition, the use of mRNA alone is suggested as the necessary endpoint to evaluate induction response. However, the applicability of using prequalified hepatocytes as the test system, and the use of mRNA alone as a response endpoint, has not been widely demonstrated. To this end, we have examined the induction response from a set of 20 model compounds which includes established clinical inducers and non‐inducers of CYP3A4/5, in cryopreserved hepatocytes from three donors. Endpoints examined were CYP3A4 mRNA and testosterone 6ß‐hydroxylase activity, typically over 8 concentration points to enable calculation of EC50 and Emax. Results were used to determine relative induction scores (RIS), R3 values, and fold‐induction threshold values that may classify a drug candidate as a potential clinical inducer. The results and merits of these and other parameters for evaluating induction potential of drug candidates will be discussed.
A novel paradigm for protection and treatment for seasonal allergy
Authors: Prayaga Sudhirdas , Venugopal Changaram
Published: June 2021

Here we detail our approach to validate our hypothesis that peptide sequence from environmental allergens may be used as potential vaccine candidate for seasonal allergy. Seasonal allergy is caused by exposure of susceptible individuals to environmental allergens, such as pollens and microbes, which causes the release of inflammatory mediators causing the symptoms of allergy. Currently there is no treatment or therapy of allergy, except controlling the symptoms of the disease. We have identified a peptide, from a native protein, against which we found significant amount of ‘auto‐antibodies’ in healthy horses compared to animals with recurrent airway obstruction (RAO), an animal model for seasonal allergy. Computational sequence analysis of peptide has revealed significant homology to various environmental allergens, including the common etiological agent Aspergillums. Further analysis of eight more healthy horses validated our hypothesis that peptide specific antibodies may be protective. Therefore, we hypothesize that active protective immunity to allergy can be generated in susceptible horses and immune response of healthy horses can be modified by proper vaccination protocol using this peptide.
Generation of high affinity anti‐methamphetamine antibodies using structure‐based engineering
Authors: Thakkar Shraddha , Nanaware‐Kharade Nisha , Gonzalez Guillermo , Celikel Reha , Peterson Eric C. , Varughese Kottayil I.
Published: June 2021

Methamphetamine (METH) abuse is a threat in the US and worldwide, with no FDA approved treatments available. Anti‐METH IgGs and its smaller variant, single chain fragments (scFvs) have shown efficacy in preclinical studies. The goal of study is to improve efficacy of a therapeutic single chain fragment (scFv6H4) by increasing its affinity for METH. Here we describe our efforts to design higher affinity antibody mutants based on the crystal structure of scFv6H4, in complex with METH. Using the structural information, we made single amino acid mutations in the binding pocket of scFv6H4 to alter the hydrophobicity of the binding site. The new mutants; scFv‐S93T, ‐I37M, and ‐Y34M were cloned, expressed in yeast and tested for affinity by saturation binding techniques. Two of the mutant scFv showed enhanced binding affinity for METH; scFvI37M by 25% and scFvS93T by 150%. We have also determined the crystal structure of the highest‐affinity mutant scFvS93T in complex with METH. This structure shows that, as we hypothesized, the substitution of a Ser residue by a Thr clearly facilitated the removal of a water molecule from the binding cavity. Interestingly, this mutation increased the hydrophobicity of the binding pocket as a result of expulsion of water molecule, and hence dramatically improved the binding of METH in the cavity. NIDA R01 DA026423 (ECP)
The Sublingual Glycocalyx : A Novel Noninvasive Predictor of Coronary Artery Disease in Females
Authors: Brands Judith , Vink Hans , Reis Steven , Pacella John
Published: June 2021

The glycocalyx (GC) provides in healthy blood vessels limited access to flowing red blood cells (RBC). Recent studies have suggested that RBCs penetrate deeper into a damaged GC, which has been associated with microcirculatory dysfunction that accompanies coronary artery disease (CAD). We hypothesize that the presence of CAD could be predicted by measuring the dimension of the RBC perfused boundary region (PBR), the space between median RBC column width and the outer edge of RBC permeable vascular lumen. In healthy vessels PBR is relatively small due to limited penetration of RBC into the GC and it is hypothesized that PBR will increase when GC is damaged. We used sidestream dark field imaging of the sublingual microvasculature in post‐menopausal female subjects with angiographic CAD (N=10) and healthy controls (N=15). From these images, PBR was determined in over 3000 vascular segments (diameter 5–30 μm) per subject. The mean PBR increased from 2.08 ± 0.07 μm to 2.34 ± 0.08 μm (P<0.05) comparing controls with CAD patients, respectively. These data demonstrate that CAD patients have a significant increase in the RBC accessible component of the sublingual microvascular GC compared to control subjects. A wider permeable portion of the GC indicates a change in GC structure which may be associated with a reduced vascular protective function of the GC. Supported by AHA 12POST9400001 and NIH K08 1 K08 HL086730–01
A Transgenic Cre Mouse Line for the Study of Kidney Pericytes and Perivascular Fibroblasts
Authors: Grgic Ivica , Hofmeister Andreas , Humphreys Benjamin
Published: June 2021

Pericytes and perivascular fibroblasts are considered a main source of myofibroblast recruitment in fibrotic kidney disease and currently there is a lack of suitable Cre driver mouse lines to study this cell population in vivo.

We generated a novel pericyte‐specific conditional Cre mouse by using the collagen1α1 promoter/enhancer to drive expression of a triple fusion protein consisting of GFP, Cre recombinase and the mutated estrogen receptor ERT2. For validation, we crossed this Coll1α1GCE mouse to several reporter lines and detected genetically labeled cells in the renal tubulointerstitium of tamoxifen‐treated bigenic offspring. In line with a pericyte identity, these cells stained positive for pericyte marker PDGFRβ but not for CD31, F4/80, CD3, αSMA or FSP1/S100A4 in healthy kidneys and displayed a close spatial association with endothelial cells. One submaximal dose of tamoxifen at P7 labeled ~1.5%, three consecutive pulses (P7‐P9) ~26.8% of all PDGFRβ+ pericytes/perivascular fibroblasts. Upon UUO surgery, fate labeled pericytes aquired αSMA positivity reflecting their differentiation into myofibroblasts. This transformation was accompanied by a notable increase in cell size and overall anatomical complexity.

In summary, we have created a novel pericyte‐specific conditional Cre driver mouse as a new tool to study the biology of pericytes in health and disease.
Stability of shunt flow pathways following thermal injury confirmed by multi‐wavelength laser speckle contrast imaging of oxy‐hemoglobin.
Authors: Frame Mary (Molly) , Dewar Anthony M , Hartig Renee , Islam Farha , Zhou NaiYun , Du Congwu
Published: June 2021

Our goal was to determine the stability of flow shunt pathways causing increased heterogeneity of flow with inflammation. Thermal injury (50C thermocouple controlled 300um spot burn) was used as a non‐pharmacological means to create a zone of inflammation with control regions in the same tissue. Intravital microscopy of the cheek pouch in male hamsters (N=10, isoflurane) was viewed. Rhodamine labeled RBC confirmed shunt pathways through all terminal arteriolar networks (NETS) within 500um of a spot burn (burn zone, paired locations before v 15m‐2hr after). Flow was redirected along arcades away from the burn zone. Oxy‐and deoxy‐hemoglobin (HbO 630nm v HbR 570nm) was measured (multi‐wavelength laser speckle contrast imaging) across a 2×2cm area containing the burn. Total Hb was consistent with tube hematocrit of 5–40%. Locations within 500um of the burn rim (near) v further away (far) show HbO was 30% less in perfused large arterioles (>;50um) near v far from the burn, and 24% less in perfused large venules near vs far. Within NETS far from the burn, HbO was 7% higher along the shunt vs the nonshunt pathway. Near the burn, HbO is 71% greater along the shunt vs the non‐shunt pathway. Comparing HbO along shunt pathways, HbO was decreased 52% near vs far from the burn rim. Data with HbO or RBC flux agreed. Thus, a diminished oxygen shunt pathway is initiated by thermal injury and maintained for several hours. AHA 0655908T
Numerical study of blood flow in a microchannel bifurcation with the large deformation model
Authors: Ye Swe Soe , Ju Meongkeun , Ng Yan Cheng , Tan Kok Soon , Kim Sangho
Published: June 2021

The present study investigated the red blood cell (RBC) strain‐stiffening behaviour in a microchannel bifurcation flow. The numerical model employed the Lattice Boltzmann Method and the Immersed Boundary Method. Vitally, a large deformation model was developed for the extensional and bending behaviour of the RBC membrane at high shear condition. Two sets of simulations were conducted for a 90° bifurcation microchannel with 55% inlet hematocrit over a range of non‐dimensional shear rate G from 0.0125 to 0.4; one simulation set employed the large deformation membrane model while the other was conducted with the Neo‐Hookean membrane model. The highly elongated RBCs simulated by the Neo‐Hookean model fail to match experimental observations of RBC shapes at the corresponding shear rates whereas the simulation set conducted with the large deformation model exhibited limited RBC deformation that is in good agreement with experimental results at high shear rates. Consequently the large deformation model predicts a lower incidence of RBCs travelling into the plasma‐rich daughter channel. In conclusion, this comparative study underlines the necessity of representing finite strain behaviour of RBCs when subjected to high shear stress present at the bifurcation zone.

This work was supported by NMRC/CBRG/0019/2012.
cAMP agonists stabilize the microvascular barrier in ischemiareperfusion
Authors: Korayem Adam H. , Kim David D. , Rana Roshniben J. , Paris‐Roncayolo Magdalena , Duran Walter N.
Published: June 2021

Compartment syndrome, caused by excessive leakage of fluid and protein from the vascular compartment into skeletal muscle, is a severe complication in ischemia‐reperfusion (I‐R) injury. While the causes of vascular hyperpermeability are well understood, the mechanisms of hyperpermeability deactivation are largely unknown. Because cAMP is linked to restoration of endothelial barrier properties, we tested the hypothesis that glucagon‐like peptide 1 (GLP‐1, a cAMP agonist) stabilizes the microvascular barrier. We used cross‐clamping of the hamster cremaster artery as an in vivo I‐R model, and determined permeability to FITC‐dextran‐150 measuring integrated optical intensity. We used confluent monolayers of human microvascular endothelial cells (HMVEC) to assess molecular changes associated with hypoxia‐reoxygenation (in vitro I‐R model). Topical application of GLP‐1 reduced the I‐R induced hyperpermeability in the hamster cremaster relative to control. Reoxygenation of HMVEC increased the levels of Rap‐1‐GTP and Rac‐1‐GTP. Interestingly, changes in Rac‐1‐GTP preceded changes in Rap‐1‐GTP indicating that cAMP may activate several molecular pathways to restore endothelial barrier properties following I‐R. We propose that cAMP increasing agonists, such as GLP‐1, will assist vascular surgeons in preventing compartment syndrome. (Supported by NIH grants 5RO1 HL070634 & 5RO1 HL088479).
Glucagon‐like Peptide‐1 (GLP‐1) Attenuates Agoniststimulated Cytosolic Free Calcium ([Ca2+]i) Signaling and the Production of F2α‐isoprostane in Endothelial Cells
Authors: Richardson Vanitra A , Weber Craig S , Lynch Ronald M , Dokken Betsy B
Published: June 2021

Endothelial dysfunction contributes to cardiovascular disease in diabetes. Glucagon‐like peptide‐1 (GLP‐1) is an incretin hormone that forms the basis of a class of drugs used to treat type 2 diabetes, but its effect on microvascular endothelium is largely unknown. F2α‐isoprostanes (F2αI) have vasoconstrictor properties and are produced in excess in diabetic endothelium. A rise in [Ca2+]i is required for the free‐radical mediated synthesis of F2αI. In the present study, the Ca2+‐sensitive dye Fura‐2 was used to evaluate the effect of GLP‐1 on the ATP‐induced [Ca2+]i rise in human cardiac microvascular endothelial cells (HMVEC‐Cs). In addition, human umbilical vein endothelial cells (HUVECs) were exposed to GLP‐1 (0.3 nM) for 48 hr and the release of F2αI into the media was determined by enzyme immunoassay (EIA). GLP‐1 depressed the ATP‐induced elevation of [Ca2+]i (109 ± 15 nM [control] vs. 44 ± 6 nM [GLP‐1], P<0.05) in HMVEC‐Cs, and also attenuated F2αI release by HUVECS (5.8 ± 0.1‐fold over baseline [control] vs. 3.6 ± 0.2‐fold over baseline [GLP‐1], P<0.05). These results suggest that GLP‐1 may improve endothelial function in diabetic patients, in part by decreasing the [Ca2+]i –dependent release of F2αI, an endothelium‐dependent constricting factor. This study was supported by the University of Arizona Sarver Heart Center, and the Harold and Nancy Willingham Foundation.
Differential impact of dilator stimuli on increased myogenic activation of cerebral and skeletal muscle resistance arterioles in obese zucker rats
Authors: Butcher Joshua T , Stanley Shyla C , Frisbee Jefferson C
Published: June 2021

The obese Zucker rat (OZR) model of the metabolic syndrome was used to determine the impact of dilator stimuli on myogenic activation of ex vivo gracilis muscle arterioles (GA) and middle cerebral arteries (MCA) to determine if elevated ROS and thromboxane A2 (TxA2) not only exacerbate myogenic constriction in GA and MCA of OZR, they also prevent the normal blunting of myogenic activation with elevated dilator stimuli. Myogenic activation was increased in GA and MCA from OZR vs. LZR, and the increased response was dependent on elevated ROS and TxA2. Both hypoxia and acetylcholine partially attenuated myogenic activation in GA and MCA from OZR, although this effect was muted vs. LZR. However, pre‐treatment with TEMPOL and/or SQ‐29548 increased the effect of hypoxia and acetylcholine, such that myogenic activation of vessels from OZR was comparable to that for LZR. Elevated adenosine blunted myogenic activation of GA and MCA of OZR to levels similar to that from LZR. These results suggest that, while enhanced myogenic activation of GA and MCA in OZR is partially a function of elevated ROS and TxA2, the influences of dilator stimuli blunting myogenic activation are also impaired as a result of similar processes. Treatment of vessels from OZR with antioxidants and/or blockers of TxA2 not only restore normal myogenic activation, they also improve dilator reactivity and the ability of dilator stimuli to override myogenic tone.
Crosstalk between NO and cAMP signaling in basilar arteries from young and old mice
Authors: Pfitzer Gabriele , Lubomirov Lubomir T. , Welter Johannes , Weckmueller Kathrin , Todorovic Nikola , Papadopoulos Symeon
Published: June 2021

The aim of this work was to elucidate whether aging affects crosstalk between NO and cAMP signaling in mouse basilar arteries. Isometric force was recorded by wire myography. In arteries from young mice inhibition of NOS with 100 μM L‐NAME increased basal tone by 9 % of Fmax (Fmax= max. contraction elicited by 1 μM U46619 + L‐NAME) and increased the pEC50 value of U46619 from 6.5 to 7.1 (n = 6). Inhibition of nNOS by 1 μM L‐NPA increased all parameters by a similar extend (n = 3). Pretreatment with L‐NAME completely reduced immunoreactivity against pMYPT1‐S695, the PKG site. In arteries from aged animals, L‐NAME increased basal tone by 23 % of Fmax (n = 4), but L‐NPA only by 11 %. In the absence of NOS inhibitors, pEC50 for U46619 was 7.0, comparable to that with L‐NAME or L‐NPA in young animals. Here, L‐NAME or L‐NPA increased pEC50 for U46619 only by 0.3 log‐units. In both age groups, urocortin, which acts through cAMP, completely relaxed L‐NAME induced basal tone and submaximally preconstricted (0.3 μM U46619 + L‐NAME) vessels. Activation of Epac by 8‐pCPT‐O’‐Me‐cAMP (8‐pCPT) relaxed tone induced by NOS inhibition by ~40 % in both age groups. L‐NAME dramatically reduced relaxation of preconstricted arteries by 8‐pCPT in aged but not in young vessels. In conclusion, our results suggest that regulation of basal tone in basilar arteries is nNOS‐dependent in young mice and involves both n‐ and eNOS in old mice. We also provide evidence that NOS‐activity is required for dilation by the cAMP effector, Epac in aged arteries. Funding: DFG
The impaired glycocalyx increases the endothelial susceptibility to shear stress resulting in augmented nitric oxide production in microvessels of diabetic rats
Authors: Xu Sulei , He Pingnian
Published: June 2021

Glycocalyx (GCX), a layer of proteoglycans covering the endothelium, has been implicated as a mechanical sensor for changes in shear stress (SS) in vitro. Though acute hyperglycemia has been reported to impair GCX in human by indirect volume estimation, the direct experimental evidence and how the impaired GCX affects SS‐mediated endothelial function in vivo remain to be demonstrated. The objective of this study is to identify the changes in GCX in microvessels of streptozotocin‐induced diabetic rats and evaluate the associated changes in sensing SS and SS‐induced nitric oxide (NO) production in individually perfused venules of diabetic rats. GCX is visualized either by electron microscope (EM) or by lectin fluorescence labeling. EC NO production was quantified in DAF‐2 DA loaded vessels using fluorescence imaging. Microvessels of diabetic rats showed degraded GCX by both EM and confocal images, a similar observation found in pronase (0.1 mg/ml, 3 min) treated microvessels. Unexpectedly, the EC NO production in response to blood flow‐generated changes in SS in diabetic vessels was significantly higher than that in normal vessels. The NO production rate in normal vessels were 0.13 ± 0.02 and 0.24 ± 0.02 AU/min at 11 and 32 dyn/cm2. In contrast, the NO production rate of diabetic vessel in response to the same magnitude changes of SS were 0.25 ± 0.05 and 0.37 ± 0.03 AU/min, respectively. These findings were also supported by an increased NO production observed in pronase treated normal venules. Our results indicate that the impaired GCX in diabetic microvessels enhances EC response to mechanical force and potentiates NO production. Supported by HL56237, HL084338 and AHAPRE11470010.
17β‐estradiol attenuates renal TNFα and the progression of hypertension in mice with systemic lupus erythematosus
Authors: Gilbert Emily Lane , Mathis Keisa W , Ryan Michael J
Published: June 2021

The autoimmune disorder systemic lupus erythematosus (SLE) primarily affects young women suggesting a pathogenic role for estrogen. Cardiovascular and renal disease are the major causes of mortality in women with SLE, and hypertension, a major cardio‐renal risk factor, is prevalent in these women. This led to the hypothesis that estrogen promotes hypertension and renal injury during SLE. To test this, 30 week old female SLE (NZBWF1) and control (NZW/LacJ) mice underwent either an ovariectomy (OVX) or sham operation. A subset of OVX mice were supplemented with 17β‐estradiol (E2). At 34 weeks of age, mean arterial pressure (MAP in mmHg) was higher in SLE sham mice (133±3) than controls (120±3, p<0.05) as was the prevalence of albuminuria (33% vs 0%). OVX did not alter MAP (115±3) or albuminuria in controls; however, contrary to our hypothesis, OVX in SLE mice exacerbated the hypertension (154±3, p<0.05 vs SLE sham) and prevalence of albuminuria (75% vs 33%). This was associated with increased renal TNFα protein expression (3.2±0.8 SLE OVX vs 1.8±0.5 SLE sham), an inflammatory cytokine that contributes to SLE hypertension. E2 repletion prevented the increase in pressure caused by OVX in SLE mice (132±2) and reduced albuminuria and renal TNFα (2.3±0.3). These data suggest that estrogen protects against SLE hypertension via reduction of renal inflammatory cytokines. (HL085907, HL051971, AHA12050150, UMC‐IRSP).
Potentiation of angiotensin II (Ang II) hypertension in rats by sensitizing pre‐treatment with intermittent ICV Ang II injections
Authors: Clayton Sarah C , Hurley Seth , Beltz Terry , Johnson Alan Kim
Published: June 2021

Previous work from our lab has shown the potentiated effect of pre‐treatment with subpressor infusion of Ang II on the hypertensive response to a pressor dose of Ang II. Other studies in the sensitization field have shown intermittent brain stimulation has an effect to increase Ang II related responses, i.e. thirst. We hypothesized that intermittent injection of subpressor Ang II would also sensitize the blood pressure (BP) response to a pressor dose of Ang II. To study this hypothesis, we instrumented rats with radiotelemetry devices to measure BP and ICV cannula for Ang II injection and employed an Induction‐Delay‐Expression (I‐D‐E) experimental design. Rats were allowed to recover for one week before baseline recordings were made (B). After B, rats were injected with 10 ng Ang II in 4 μl ICV on days 1, 3, 5, 7 (I). Rats were then rested an additional 7 days (D); after D, rats were implanted with osmotic minipumps to deliver Ang II (120 ng/kg/min, sc) continuously for 2 weeks (E). No difference in BP was detected during B, I, or D in either group. BP increased during E in both groups (sham: 111±3 mmHg, B, vs. 126±3 mmHg, E; pre‐Ang II: 105±4 mmHg, B, vs. 140±7 mmHg, E); however, this increase was potentiated in rats with intermittent Ang II injections (delta: 15±5 mmHg, sham, vs. 35±5, pre‐Ang II, P=0.03). These results document the ability of central intermittent Ang II injections to sensitize the BP response to systemic Ang II.
Role of Prostaglandin D Synthase in Cardiac Remodeling Secondary to Pressure Overload in Rats
Authors: Nagalla Krishna T , Gole Monica Sharad , Murray David B
Published: June 2021

We sought to compare the effects of specific COX 2 inhibitors (Nimesulide) or Prostaglandin D synthase inhibitor (HQL 79) on cardiac remodeling secondary to pressure overload in rats. In vivo left ventricular (LV) structure and function was assessed by pressure/volume catheter at 28 days post surgery in six groups (n ≥ 6 per group): sham‐operated (Sham); untreated pressure overload (PO); Prevention or intervention groups treated with either NIMEsulide (25 mg/kg/d) or HQL 79 (10 mg/kg/d). The prevention strategy was initiated prior to induction of pressure overload and continued to day 14. Intervention treatment strategy started on day 14 post induction of pressure overload and continued till day 28. Both prevention and intervention NIME treatments attenuated the PO induced change in LV mass. Prevention treatment with HQL 79 attenuated this change. End diastolic volume was significantly decreased (by 29%) in PO group compared to Sham. Both treatment strategies with HQL 79 significantly attenuate this change. Prevention treatment with Nimesulide significantly attenuated the change in PO induced increase in total collagen levels, whereas both prevention and intervention treatment with HQL 79 significantly attenuated fibrosis. These findings indicate that cardiac remodeling during pressure overload occurs by Prostaglandin D2 mediated inflammatory pathway and can be attenuated using PGD synthase inhibitors.
Changes in circulating levels of AMP Kinase (AMPK) isoforms are associated with kidney dysfunction in hypertension
Authors: Jian Ming‐Yuan , Creighton Judy
Published: June 2021

AMPK is a metabolic sensor and master regulator of homeostasis at all physiologic levels. We previously demonstrated that loss of AMPKα was associated with abnormal vasculature in the hypertensive lung. In this study, we sought to determine whether altered expression patterns could be monitored in vivo using non‐invasive methods. Changes in the expression patterns of specific AMPK α,β,γ isoforms were detected in the blood of spontaneously hypertensive rats as they aged and kidney function declined. In particular, loss of AMPKα1, AMPKα2 and increased levels of AMPKβ1 were detected by PCR and western blot analysis in the serum of aged animals. These changes correlated with increased lactate, and BUN levels and changes in morphology of the kidney classically associated with hypertension (e.g. glomerulosclerosis, tubular atrophy, interstitial fibrosis, and arterial intimal hyalinosis). In hypertensive kidneys, loss of AMPKα1 and AMPKα2 was noted throughout the organ, whereas AMPKβ1 was increased in the distal tubules. These data identify AMPK as a potential biomarker of kidney dysfunction and clinical tool for accessing disease progression in hypertensive patients. Supported by HL102296 and HL 110803.
Maternal separation (MS) increases acute and chronic norepinephrine (NE) sensitivity revealing sympatho‐activation
Authors: Loria Analia Silvina , Hokanson Monica , Pollock David M , Pollock Jennifer S
Published: June 2021

We reported that MS, a model of early life stress (ELS), induces a greater drop in blood pressure in response to a ganglion blockade and a reduction in the renal filtration capacity that was normalized by renal denervation. We hypothesized that MS sensitizes the peripheral sympathetic system. The aims of this study were to evaluate 1) NE content in vasculature, kidney, spleen, and adrenal tissues, and 2) vascular tone in response to adrenergic agonists from 12 week old MS and control WKY rats. MS consisted of separating half of the male pups from the dam 3 hr a day from days 2–14 of life. NE was unchanged in plasma, thoracic and abdominal aorta while NE content was significantly increased in renal cortex (331±26 vs. 219±38 ng/g tissue), spleen (473±66 vs. 253±28 ng/g tissue) and adrenal gland (1152±189 vs. 303±99 vs. ng/g tissue) from MS vs. control rats (p<0.05). Rats were injected with NE (2 μg/kg, i.p.) and blood pressure monitored by telemetry. Mean arterial pressure was exacerbated in MS vs. control rats (178±1 vs. 147±10 mmHg, p<0.05) within 15 minutes. In a separate group of anesthetized rats, reduction in renal blood flow in response to phenylephrine was attenuated in MS rats compared to control rats (−1.8±0.2 vs. −2.8±0.2 Δml/min, p<0.05, 100 ug/kg), suggesting an adrenergic desensitization. Thus, ELS sensitizes the sympathetic system impairing the normal acute and chronic vascular tone regulation.
Endothelin B (ETB) receptor protects against endoplasmic reticulum (ER) stress‐induced renal damage
Authors: De Miguel Carmen , Hobbs Janet L , Pollock David M , Pollock Jennifer S
Published: June 2021

Tunicamycin (TM) blocks glycoprotein synthesis, leading to endoplasmic reticulum (ER) stress and tissue damage. Endothelin (ET1) via ETA and ETB receptors is implicated in renal injury. We hypothesized that ETB deficient rats (sl/sl) develop exaggerated renal ER stress and/or inflammation compared to wild type (wt) rats in response to acute TM treatment. Wt and sl/sl rats (n=3–5) were treated with TM (2μg/g bwt; i.p.) or vehicle; kidneys, plasma and urine were collected 24h later. ER stress markers and cytokines were determined by qRT‐PCR in renal cortex, outer and inner medulla. Plasma, urine ET1 and renal inflammatory cell infiltration were assessed by ELISA and histology. Sl/sl rats showed enhanced sensitivity to TM by increased expression of GRP78 in inner medulla (wt vs. sl/sl, fold change: 1.52±0.31 vs. 4.60±0.32) and outer medulla (153.6±57.3 vs. 3413±625.8), CHOP in inner medulla (1.46±0.63 vs. 25.50±2.6) and cortex (0.34±0.08 vs. 12.08±2.77), and spliced XBP‐1 in outer medulla (0.91±0.61 vs. 15.71±1.92) and cortex (0.28±0.03 vs. 7.96±1.84). TM did not increase plasma ET1 or urinary ET1 excretion in either rat genotype. Renal CCL2 and IL‐6 expression, as well as macrophage and T‐cell infiltration, were similar between both genotypes in response to TM. Induction of renal ER stress genes is significantly greater in sl/sl rats, thus ETB receptors may be protective against ER stress‐induced renal damage.
T cells mediate angiotensin II‐induced aortic stiffening
Authors: Wu Jing , Thabet Salim R. , Kirabo Annet , Trott Daniel W. , Goldstein Anna , Hudson Billy G , Madhur Meena S. , Chen Wei , Harrison David G.
Published: June 2021

In this study, we sought to determine if T cells mediate aortic stiffening in hypertension. Histological studies showed that chronic angiotensin II infusion caused profound collagen deposition in the aortic adventitia of wild‐type (WT), but not lymphocyte deficient (RAG‐1−/−) mice. Biochemical analysis confirmed that angiotensin II increased collagen from 3.3±0.02 to 6.6±0.5 μg/mm (sham vs ang II, p<0.01, n=5) in WT mice but not in Rag‐1−/− mice. Total elastin was neither different between mouse strains nor changed by angiotensin II. Angiotensin II decreased aortic compliance (measured in isolated pressured vessels) in WT mice by 60% (p<0.01, n=8), but not in Rag‐1−/− mice. Adoptive transfer of pan T cells into Rag‐1−/− mice restored the aortic stiffening and collagen deposition caused by angiotensin II. Studies with interferon gamma (IFN‐γ) and IL‐17A deficient mice indicated that both of these cytokines contribute to aortic collagen deposition and stiffening. In conclusion, T cells are essential for angiotensin II‐induced collagen deposition and aortic stiffening. Part of these effects are mediated by T cell‐derived cytokines such as IFN‐γ and IL‐17A.
Processing of Angiotensinogen to Angiotensin‐(1–12) by a Non‐ Renin Enzyme in the Salt‐Sensitive mRen2.Lewis Rat
Authors: Americo Anna , Pirro Nancy , Brosnihan K. Bridget , Chappell Mark
Published: June 2021

The mRen2.Lewis (mRen2) congenic rats exhibit a marked salt‐sensitive response in regards to blood pressure, oxidative stress, and proteinuria. The expression of renin within the kidney of the mRen2 is suppressed which likely reflects the intact response to the higher blood pressure; however, the urinary excretion of angiotensinogen (Aogen), Ang II and Ang‐(1–7) is significantly increased in high salt (HS) fed mRen2. The present study examined the renal response to a HS diet (4% Na) in female mRen2 regarding the urinary components of the renal RAS. In comparison to normal salt (0.5% Na), the HS diet markedly increased the urinary excretion of Aogen [1.8 ± 0.2 vs. 0.01 ± 0.001 μg/kg/day, p<0.01] and Ang‐(1–12) [4.6 ± 1.1 vs. 164 ± 21 pmol/kg/day; p<0.01]; however, Ang I excretion was not changed. Pooled and concentrated samples from the HS mRen2 were incubated with Aogen substrate (pH 7.0, 37ºC) and the generation of Ang‐(1–12) and Ang I determined by separate RIAs. Ang‐(1–12) was the predominant product from Aogen [1.2 pmols/min] as compared to Ang I [0.05 pmols/min]. HPLC analysis of the enzyme assay revealed a single immunoreactive peak for Ang‐(1–12). Although Ang I generation was abolished by the renin inhibitor aliskserin, the formation of Ang‐(1–12) was slightly enhanced. We conclude that the processing of Aogen to Ang‐(1–12) in the salt‐sensitive mRen2 likely reflects a renin‐independent pathway within the kidney.
Relationship between renal Ang (1–7) induced sodium and water excretion and AT1 receptors.
Authors: Neill Julie O , Healy Vincent , Johns Edward J
Published: June 2021

This study investigated the relationship between renal excretory actions of Ang (1–7) and the AT1 receptor. Male Wistar rats were fed a normal (0.3%) or high (3%) sodium diet for 2 weeks. Rats were anaesthetized (chloralose/urethane) and either prepared for left renal function measurement and intrarenal (IR) infusion of drugs or both kidneys were removed and prepared for measurement of cortical AT1 receptor expression using Western Blotting. Data ±SEM were subjected to a Students t‐test and significance set at (P<0.05). Mean arterial pressure (MAP), Fractional Sodium Excretion (FENa) and Urine Volume (UV) averaged 94±4mmHg, 1.1±0.3%, and 33.7±10.3 μL/min/Kg respectively across both dietary groups. Ang (1–7) increased UV by 160% and FENa by 227% in normal sodium fed rats (both, P<0.05). In high sodium fed rats and during IR AT1 receptor blockade (Losartan), Ang (1–7) induced increases in UV and FENa were blunted at 64% and 87% and at 6% and 19% (both P<0.05) respectively. There was also a 2 fold reduction (P<0.05) in cortical AT1 receptor expression in high sodium fed rats. These data indicate that the renal excretory actions of Ang (1–7) may be dependent upon AT1 receptor functionality and expression.
Acute changes in glomerular albumin filtration during systemic infusion of angiotensin II – a multiphoton microscopy study
Authors: Schiessl Ina Maria , Castrop Hayo
Published: June 2021

Antiproteinuric effects of ACE inhibitors have classically been assigned to their hemodynamic effects. Here we assess the acute effects of angiotensin II (AngII) on albumin glomerular sieving coefficient (GSC) in young anesthetized Munich Wister Froemter rats using intravital microscopy. Alexa‐Fluor‐594 albumin was injected i.v. and fluorescence intensities were determined in glomerular capillaries and in Bowman's space (BS). The albumin GSC was then calculated according to: GSC = (intensity BS – intensity background)/(intensity capillaries – intensity background). GSC was measured before and during constant infusion of Ang II (27 ng/min/g BW). Baseline MAP averaged 90±4 mm Hg and stabilized at 130+/−11 mm Hg during AngII infusion. Capillary flow velocity was reduced during AngII infusion. Albumin GSC averaged 0.00038+/−.00009 at baseline and increased by 263+/−34% within 10 min after constant AngII infusion. During AngII infusion, de novo fluorescence was observed in the proximal tubules indicating uptake of filtered Alexa‐albumin. These effects were abolished when a bolus of the AT1 antagonist losartan (9.9 μg/g BW) was injected prior to AngII. In control experiments (saline infusion) no significant changes in MAP, GSC or tubular fluorescence were observed. In summary, AngII causes rapid increases in glomerular albumin filtration, which are presumably mediated by angiotensin AT1 receptors.