bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced catecholamine sensitivity is related to a decrease in adenylate cyclase activity mediated by ß1‐adrenergic receptor signaling in aged ventricular myocytes
Authors: Farrell Spring RM , Howlett Susan E
Published: June 2021

The aim of this study was to determine whether reduced sensitivity to catecholamines in aged myocytes results from deficits in ß‐adrenergic receptor (AR) signaling. Contraction amplitudes and intracellular Ca2+ were measured in field‐stimulated (2 Hz, 37°C, fura‐2) ventricular myocytes isolated from young adult (3 mos) and aged (24 mos) male Fischer 344 rats. The effect of a ß1‐AR agonist (but not a ß2‐AR agonist) on contraction amplitude was greater in young adult compared to aged myocytes; however, Ca2+ transient amplitudes were similar in both groups. To examine downstream effectors of ß‐AR signaling we assessed the activity of adenylate cyclase and the formation of cAMP. Aged myocytes had smaller increases in contraction and Ca2+ transient amplitudes in response to forskolin compared to young adult cells. In addition, direct measurement of cAMP showed an age‐related decrease in cAMP formation in response to forskolin. This reduction in adenylate cyclase activity is likely responsible for the age‐related decrease in catecholamine sensitivity as administration of dibutyryl cAMP showed no age bias on contraction or Ca2+ transient amplitude. Funding: CIHR & HSFNS.
Nitric oxide modulation of central imidazoline I1 receptors accounts for the hypertensive effect of cyclosporine in rats
Authors: El‐Mas Mahmoud M , Omar Amal G , Helmy Mai M , El‐Din Mahmoud M Mohy
Published: June 2021

This study tested the hypothesis that the hypertensive action of cyclosporine (CSA) involves interaction with central α2‐receptors and/or I1‐imidazoline receptors and investigated the role of nitric oxide in the interaction. The effect of clonidine (mixed α2/I1‐receptor agonist), moxonidine (I1‐receptor agonist), or guanabenz (α2‐receptor agonist) on CSA hypertension was assessed in conscious rats. CSA (20 mg/kg i.v.) increased BP that continued for at least 45 min. CSA hypertension was inhibited by ganglionic (hexamethonium) or α1‐receptor (prazosin) blockade. Pressor responses to phenylephrine were not affected by CSA, thereby eliminating a possible role for α1‐receptors in CSA hypertension. Intravenous guanabenz (30 μg/kg), clonidine (30 μg/kg), or moxonidine (100 μg/kg) produced similar decreases in BP. Pretreatment with clonidine or moxonidine attenuated CSA hypertension whereas guanabenz had a minimal effect, suggesting a predominant role for I1‐receptor neuronal pathways in the BP effect of CSA. Central administration of moxonidine (80 nmol) into cisterna magna also attenuated CSA hypertension. NOS inhibition by NG‐nitro‐L‐arginine methyl ester abolished BP responses to moxonidine and CSA.

Conclusion: facilitation of central I1‐receptor‐NO signaling accounts, at least partly, for CSA hypertension.

Supported by Faculty of Pharmacy, Univ. of Alexandria, Egypt.
Statin‐induced Myotoxicity: Update on Case Characteristics and Recruitment Efficiency in a Clinical Practice‐based Setting
Authors: Mareedu Ravi K , Modhia Falgun M , Krauss Ronald M , McCarty Catherine A , Wilke Russell A
Published: June 2021

Objectives: To characterize the clinical characteristics of the 181 research subjects participating in a retrospective case‐control study of the genetic factors underlying statin‐induced myotoxicity.

Methods: Medical records of approximately 2,000,000 unique patients in a multi‐specialty group practice were interrogated electronically to identify individuals who had a serum creatinine kinase (CK) level drawn and at least one clinical note with text mentioning either atorvastatin, or, simvastatin, or, pravastatin, This yielded approximately 27,450 unique subject records. Inclusion of the upper 10th percentile for CK level (>370 IU/dL) resulted in 2745 patients. Eligibility was determined by prior physician notes reflecting an increased index of suspicion for drug intolerance.

Results: Of 248 patients who met inclusion criteria, 181 subjects have chosen to enroll in the study. During physician review of these 181 cases (80 atorvastatin, 42 pravastatin, and 59 simvastatin), the clinical pain syndrome was graded using binary variables: 85/181 had diffuse pain, 52/181 had proximal pain and 36/181 had severe pain. 29/181 claimed recent vigorous physical exertion, and 20/181 reported the concomitant gemfibrozil use.

Conclusions: A large subset of the validated cases had either proximal pain, diffuse pain, or severe pain. (Funding U01HL069757‐06)
The Effect of PEGylated Fibrin Injection On The Recovery Of Skeletal Muscle Function Following Ischemia‐Reperfusion Injury.
Authors: Gokhale Rohit , Fegan Kelly , Suggs Laura , Farrar Roger
Published: June 2021

Ischemia/Reperfusion (I/R) injury can cause irreversible damage to muscle tissue. PEGylated Fibrin is an inert substance which can be used to deliver growth factors to improve regeneration in such irreversibly damaged muscle. In this study we evaluated the effect of PEGylated Fibrin injection on the recovery of skeletal muscles following the application of 4‐hour pneumatic tourniquets. There was no significant difference in force recovery between injection and non‐injection groups for the In Vivo force assessment (p=0.19), the In Situ force assessment (p=0.12) and the Specific tetanic tension of the muscle tissue (p=0.51). Histological assessment of the muscles did not reveal any significant differences between the injected and non‐injected muscle. While fibrin injection is known to induce an inflammatory response, it did not appear to affect recovery of function in these muscles. These results indicate that PEGylated fibrin gel has no detrimental effect on muscle tissue when injected, making it an appropriate material to deliver growth factors into skeletal muscle. This work was funded in part by the U.S. Army Medical Research and Material Command grant DAMD W81XWH‐06‐1‐0540 to RPF.
An investigative pharmacology study of a GDF‐8 (myostatin) inhibitor, ACE‐031, in the Common Marmoset (Callithrix Jacchus)
Authors: Pearsall Scott , Wachtman Lynn , Mansfield Keith , Lane Joan , Seehra Jasbir
Published: June 2021

ACE‐031 is a biotherapeutic based on the activin receptor type IIB under investigation for treatment of diseases involving loss of muscle mass, strength and function. ACE‐031 has demonstrated increased skeletal muscle mass and strength in rodent models of ALS and muscular dystrophy. The purpose of this study was to evaluate the pharmacologic effects of ACE‐031 administered once weekly for 13 weeks to marmosets via subcutaneous injection. Marmosets (n=6/sex) were randomized into groups receiving 3 mg/kg of ACE‐031 or vehicle. Body composition analysis using Dual Energy X‐ray Absorptiometry (DXA) or EchoMRITM was performed at baseline then twice monthly for the duration of the study. DEXA scans were performed with a Lunar scanner for calculation of regional body composition. Whole body composition analysis was performed using EchoMRITM technology. Animals were euthanized following last dose and subjected to complete histopathology evaluation. Skeletal muscle was subjected to fiber type analysis. Preliminary data suggest that ACE‐031 demonstrates pharmacologic changes in body composition in the marmoset, consistent with that observed in rodents. These data provide a basis for future functional studies in this species. Demonstration of activity in nonhuman primates greatly enhances our confidence in the clinical potential of this product, thereby offering hope for otherwise untreatable conditions.
Hypertonic saline solution (7,5% NaCl) in Acute Pancreatitis: the effect in hepatic expression of MMPs, HSPs and oxidative stress
Authors: Rios Ester Correia Sarmento , Moretti Ana Iochabel Soares , Soriano Francisco Garcia , Souza Heraldo Possolo , Velasco Irineu Tadeu , Szabo Csaba
Published: June 2021

To investigate if hypertonic solution (HS) reduces the development of hepatic injury in acute pancreatitis (AP).AP was induced in male Wistar rats by retrograde injection of sodium taurocholate (2.5%) into the biliopancreatic duct. Volume resuscitation was administered 1h after AP via internal jugular vein. Animals were randomized into four groups: C‐ control; APH‐ AP and treatment with HS; APS– AP and treatment with normal saline (0,9% NaCl 34 ml/Kg); APNT– AP no treatment. The animals were sacrified 4, 12 and 24h after AP induction to obtain liver samples. IL‐10 and IL‐1b, NO2/NO3, Lipid Peroxidation, HSPs and MMPs expression and MMPs activity were analyzed. Data were analyzed by ANOVA, p<0.05 were considered significant. NO2/NO3 and TBARs levels increased in APS after 12 hours, however in the APH occur an increase of NO2/NO3 at 24h. MMP9 expression and activity increased in APS at 4 and 12h, but HS treatment keeps basal levels. There is a negative correlation between NO2/NO3 and MMP9 in 24h (R=0.76 p<0.01). HS keeps MMP2 levels. HSP60 expression decreased in APH at 4h (positive correlation with MMP9 expression, R=0.86 p<0.001). HSP90 expression decreased in APS and APH at 4h. IL‐1b increased in APS at 4h and IL‐10 increased in 24h. HS modulate inflammatory response and hepatic remodeling in pancreatitis.
Efficacious treatment of alcoholic steatosis by a pharmaceutical composition comprising metadoxine and garlic oil
Authors: Kim Sang Geon , Ki Sung Hwan
Published: June 2021

Alcoholic steatosis is the earliest and common response to heavy alcohol intake, and may precede severe forms of liver injury. As an effort to develop an efficacious pharmaceutical composition, this study evaluated the effects of metadoxine, garlic oil or their combination on alcoholic steatosis. Feeding rats an alcohol‐containing diet for 4 weeks increased hepatic triglyceride content with CYP2E1 induction. Concurrent administration of metadoxine and garlic oil (MG, 1:1 w/w ratio) to rats synergistically abrogated both fat accumulation and CYP2E1 induction compared to the individual treatment. Histopathology confirmed the ability of MG combination to inhibit lipid accumulation. Blood biochemistry verified improvement of liver function in MG‐treated rats. Alcohol administration resulted in decreases in the AMPKα and ΑCC phosphorylations, which was restored by MG treatments. Hepatic fatty acid synthase (FAS) expression was decreased after alcohol consumption, which correlated with a decrease in AMPK activity and a commensurate increase in lipid content. Combined MG treatments allowed FAS expression restored. These results demonstrate that a pharmaceutical composition comprising MG effectively treats alcoholic steatosis, which may be associated with CYP2E1 repression and recovery of AMPK activity, promising pharmaceutical therapy for alcoholic steatosis.

Supported by a contract fund from the Seoul R&BD Program (2005–2010).
Curcumin reverses attenuated carbachol‐induced contraction of the colon in a rat model of colitis
Authors: Lubbard Asmaa , Oriowo Mabayoje , Khan Islam
Published: June 2021

Colitis is associated with dysmotility and reduced responsiveness of the colon to contractile agonists. Recent reports have shown that curcumin, the active constituent of Curcuma longa, was effective against experimentally induced colitis in rodents. These studies showed that curcumin prevented mucosal damage and diarrhea. The main objective of this study was to investigate the effect of curcumin on carbachol‐induced contraction of colon segments from rats with TNBS‐induced colitis. MPO activity in colon segments from TNBS‐treated rats (17.65 ± 5.43 units/min/mg tissue) was greater than in segments from control rats (0.40 ± 0.33 units/min/mg tissue). The maximum response was significantly reduced in colitic (27.2 ± 5.8 mg/mg tissue weight) compared to non‐colitic (58.0 ± 3.9 mg/mg tissue weight) rats. Carbachol produced concentration‐dependent contraction of colon segments from TNBS‐treated rats with or without treatment with curcumin. There was no significant difference in the potency of carbachol in colon segments from TNBS‐treated rats with or without curcumin treatment. In addition, there was no significant deference in the maximum response to carbachol between control rats (58.0 ± 3.9 mg/mg tissue weight, n=4) and TNBS‐rats (63.1 ± 6.6 mg/mg tissue weight, n=4) treated with curcumin indicating reversal of impaired contractility.
Fas targeted siRNA markedly attenuates sulfur mustard‐induced apoptosis in human airway epithelial cells
Authors: Keyser Brian , Andres Devon , Benton Betty , Ray Radharaman
Published: June 2021

Sulfur mustard (SM), known as mustard gas, causes debilitating skin blisters and injuries to the eyes and the respiratory tract; these effects are attributed to SM‐induced epithelial damage, mainly basal cell apoptosis. Previous studies have shown that SM‐induced apoptosis is mediated by caspases‐8 (death receptor pathway) and ‐9 (mitochondrial pathway). Our earlier results have suggested the Fas receptor is the major activator of caspase‐8 in human bronchial/tracheal epithelial cells (NHBE, upper lung model). To elucidate the role that Fas plays in SM‐induced apoptosis, we designed a siRNA construct targeted towards the Fas receptor. By utilizing siRNA technology we are able to block the Fas receptor more selectively than with other methods (e.g., by using a Fas antagonistic antibody ZB4). Since caspase‐8 activates the executioner caspase‐3, we studied Fas activation by measuring caspase‐3 using a fluorogenic caspase substrate hydrolysis assay. Validation of the siRNA construct was at both protein (western blot for Fas) and functional (caspase‐3 activation) levels. We found that siRNA was able to substantially (∼50%) decrease the SM‐induced apoptosis. These results suggest a high degree of involvement of Fas receptor in SM‐induced apoptosis. Research supported by JSTO‐DTRA Award # M0005_04_RC_C to R. Ray. The views represented are those of the authors and are not necessarily endorsed by the US Army.
SITE SPECIFIC PHOSPHORYLATION OF HEAT SHOCK PROTEIN 27 (HSP27) REGULATES CELL SURVIVAL AND DEATH
Authors: Lord‐Garcia Jean , Labenski Matthew T , Cromey Doug , Xie Ruiyu , Galligan Michael , Monks Terrence J , Lau Serrine S
Published: June 2021

Hsp27 is constitutively expressed and increases in response to stress. Pretreatment of LLC‐PK1 cells with 11‐deoxy‐16,16‐dimethyl PGE2 (DDM‐PGE2) selectively increases constitutive Hsp27 expression and contributes to protection against 2,3,5‐tris(glutathion‐S‐yl)hydroquinone (TGHQ) induced cytotoxicity. We have now identified specific changes in Hsp27 phosphorylation during DDM‐PGE2 mediated cytoprotection. 2D gel electrophoresis revealed that in addition to constitutive pS15‐Hsp27, TGHQ induces the formation of 2 additional Hsp27‐positive proteins. 2D western analysis subsequently identified TGHQ induced pS84‐ and pS88‐Hsp27, both of which are translocated to the nucleus. Pretreatment of LLC‐PK1 cells with DDM‐PGE2 increases the constitutive expression of pS15‐Hsp27 in the cytosol and the nucleus, and specifically decreased TGHQ‐mediated induction of pS84‐Hsp27. These findings were confirmed by mass spectrometric analysis. Interestingly, although the inhibition of p38 MAPK prevented TGHQ‐induced pS84‐Hsp27 phosphorylation and cytotoxicity, transfection of cells with mutant Hsp27 (S15,84,88→ A15,84,88) or p‐Hsp27 (S15,84,88→D15,84,88) had no effect on TGHQ‐induced cytotoxicity. The data suggest that the specific phosphorylation of Hsp27 at Ser84 negatively regulates cell survival, and that pS15‐Hsp27 may be positively associated with cell survival. (GM56321, ES06694).
Osmotic increase suppresses endothelial dependent vasodilation in murine cremaster m.
Authors: Georgi Melissa KB , Frame Mary D
Published: June 2021

A hallmark of metabolic disorder is endothelial dysfunction, e.g. lack of dilation to acetylcholine (Ach). We hypothesized that acute elevated glucose alone would reduce endothelial dependent dilation response, yet not effect dilation to exogenous nitric oxide (sodium nitroprusside, SNP). The cremaster muscle of anesthetized (pentobarbital 50 mg/kg) C57BL/6 mice (n=37, 103±18 d, 28±2g) was prepared for intravital microscopy to observe terminal arteriolar diameter. Ach (10‐4 – 10‐9 mM) or SNP (10‐3 – 10‐7) was applied locally via micropipette, before (0 glucose) and after 2 hour continuous exposure to suffusate glucose (15 mM, 270 mg/dl). Responses are reported as change in diameter normalized to maximum (adenosine 10 4 mMol). Maximal dilation with Ach was suppressed with glucose (0.06 ± 0.03) vs. time control (0.35 ± .05). In contrast, maximal dilation with SNP was not different with glucose (0.40 ± .03) vs. time control (0.33 ± 0.04). Mannitol (osmotic control, 15 mM) had no effect on SNP dilation (0.21±.06), yet suppressed Ach dilation (0.02±0.03). Comparing 2 hrs of control suffusate exposure with 2 hours of elevated glucose exposure, the vasoactive response to endothelial dependent dilation is affected by transient diabetic levels of interstitial glucose, while endothelium independent dilation is not. The suppression of Ach response may have osmotic origins. (NIH HL55492, DK68401)
Dilator NO, prostaglandins (PGs) and constrictor PGH2/thromboxane A2 mediate flow‐induced dilation of venules.
Authors: Racz Anita , Veresh Zoltan , Koller Akos
Published: June 2021

Mediation of flow dependent responses of venules is still not well characterized. We hypothesized that nitric oxide (NO) and prostanoids contribute to the mediation of responses. Thus, changes in diameter of pressurized venules (active diam.: 259±11 μm, and passive diam.: 412±11 μm, at 10 mmHg) isolated from rat gracilis muscle to increases in perfusate flow were measured, before and after incubation of the vessels with the prostaglandin H2/thromboxane A2 (PGH2/TXA2) receptor blocker SQ 29,548, or the NO synthase inhibitor Nω‐nitro‐L‐arginine‐methyl‐esther (L‐NAME), or the non‐specific cyclooxygenase (COX‐1 and COX‐2) inhibitor indomethacin (INDO), or the specific COX‐1 inhibitor SC 560, or COX‐2 inhibitor NS 398, or with their combination. Increases in flow elicited dilations (max. ΔD: 18±3 %), which were significantly augmented in the presence of SQ 29,548 (37±3 %). Additional presence of L‐NAME, or INDO significantly decreased the dilations (max. ΔD: 23±4 and 15±5, respectively), whereas SC 560 significantly reduced the dilations (max. ΔD: 12±6 %), and NS 398 was without effect. Thus, in isolated skeletal muscle venules, the final response to increases in flow is dilation, due to the combined effect of dilator NO and COX‐1‐derived PGE2/PGI2 and constrictor PGH2/TXA2. We propose that flow‐induced release of dilator and constrictor mediators contribute to the regulation of peripheral venular resistance. (Grants: Hungarian Sci. Res. Funds/OTKA: T48376, T767984 and Health Sci. Council/ETT 364/2006; and Am. Heart Assoc. NE Aff. 0555897T, USA)
VESGEN/PIV Analysis of Blood Flow Determined by Microvascular Branching
Authors: Parsons‐Wingerter Patricia A. , McKay Terri L. , Vickerman Mary B. , Wernet Mark P , Radhakrishnan Krishnan
Published: June 2021

Regulation of blood flow by microvascular branching was investigated by applying VESGEN 2D software (for Generational Analysis of Vessel Branching) to velocity flow fields imaged intravitally and analyzed by particle imaging velocimetry (PIV). Red blood cells (RBCs) were tracked by PIV as moving particles within capillaries, post‐capillary venules and small veins of the avian yolk sac at embryonic day 5. By newly automated, user‐interactive VESGEN 2D analysis, maximum flow velocity, volumetric flowrate and flow pulsatility obtained from highly parabolic laminar flow profiles decreased with decreasing vessel diameter and increasing branching order. In one flow field, for example, maximum and minimum average velocities of the largest (parent) vessel of generation one (G1, average vessel diameter Dv = 108 micron) were 916 and 830 micron/sec, compared to 800 and 698 micron/sec for G2 vessels (Dv = 88 micron), and 313 and 280 micron/sec for smaller vessels (lumped together as G3–G9, Dv = 18 micron). Overall, maximum average flow velocities decreased from approximately 2000 micron/sec in larger veins by 20‐fold in capillaries (NASA Glenn IR&D 04‐54).
Mapping of oxygen saturation in murine lung during intravital microscopy
Authors: Styp‐Rekowska Beata , Tabuchi Arata , Kübler Wolfgang , Pries Axel Radlach
Published: June 2021

To investigate the distribution of oxygen in the pulmonary microcirculation in vivo, we developed a murine model of pulmonary intravital microscopy combined with multispectral imaging of hemoglobin saturation (SO2). A circular window(D 7‐10μm) was surgically excised from the right thoracic wall for microscopic access to the surface of the upper right lobe of the lung. Removal of intrathoracic air via a transdiaphragmal intrapleural catheter coupled the lung surface to the window membrane. An intravital microscope was modified for epiillumination by a light guide from a monochromatizing device (Till Photonics). Mice were ventilated at 60 breath/min and images were acquired during the expiratory plateau phases. Stacks of images at preselected wavelength ranges and exposure times were analyzed with a dedicated software (SO2 Analysis Program, SOAP) to obtain 2D SO2 images of the observed area. On SO2 images, arterioles, venules and capillaries could be distinguished clearly. Results show an increase in oxygen saturation from 70% to 100% during the passage from arterioles to capillaries over a vessel length of about 200μm.
Ca2+‐activated K+ channels are controlled by Ca2+ influx through voltage‐gated Ca2+ channels, not the release of Ca2+ through ryanodine receptors in arteriolar smooth muscle
Authors: Boerman Erika Mary , Jackson William F.
Published: June 2021

Ca2+ from ryanodine receptors (RYR) does not control the activity of Ca2+‐activated K+ channels (BKCa) in microvascular smooth muscle (μVSM), unlike other systems. To further test this hypothesis, hamster cremaster arterioles were isolated, cannulated, pressurized to 80 cm H2O, loaded with Fluo 4 and confocally imaged. In vessels with myogenic tone under control conditions, we saw no Ca2+ sparks, but Ca2+ flashes were seen in 39% of μVSM (n = 198). Phenylephrine (1 μM) increased sparks (9%) and flashes (88%) (n = 207, p < 0.05). To test if Ca2+ entry through voltage‐gated Ca2+ channels (VGCC) controls BKCa, we measured diameter responses at 20 cm H2O to the BKCa antagonist, tetraethylammonium (TEA, 1 mM) in the absence and presence of a VGCC agonist, BayK 8644 (~5 nM). TEA alone caused only a 2 ± 1 μm constriction. BayK constricted vessels from 50 ± 2 μm to 38 ± 2 μm (p < 0.05). With BayK, TEA constrictions increased to 16 ± 1 μm (n = 8, p < 0.05). In Fluo 4 loaded vessels at 20 cm H2O, no Ca2+ sparks were observed, and flashes were seen in only 25% of μVSM (n = 163). In the presence of BayK, a few sparks were observed (3%), but flashes increased substantially to 87% (n = 177, p < 0.05) along with a 55% increase in global Fluo 4 intensity. Our data indicate that the activity of BKCa are not coupled to RYR in μVSM. Instead, our findings support the hypothesis that Ca2+ entry through VGCC controls BKCa activity in arterioles. Supported by PHS grant HL32469 to WFJ.
Advancing age and loss of ovarian hormones impair flow‐induced dilation in the female coronary microvasculature
Authors: LeBlanc Amanda Jo , Reyes Rafael , Muller‐Delp Judy M
Published: June 2021

Prior to menopause, the progression of cardiovascular disease with age is more rapid in men than in women; however, it remains unknown whether the cardiovascular protection experienced by premenopausal women is due to the presence of ovarian hormones. We tested the hypothesis that endothelium‐dependent dilation is impaired with advancing age in coronary arterioles from female rats, and would be further impaired by a decline in or removal of estrogen. Coronary arterioles were isolated from young (6 mo), middle‐aged (14 mo), and old (24 mo) control, ovariectomized (OVX), and OVX + estrogen‐replacement (OVE) Fischer‐344 female rats to assess dilation to flow (FID) and dea‐NONO‐ate. FID was greater in arterioles from young and middle‐aged control rats as compared to old control rats. OVX decreased FID in arterioles from young and middle‐aged rats, but had no effect in aged rats. Conversely, OVE restored or improved FID in arterioles from OVX rats as compared to control rats. Protein and mRNA expression of AKT and eNOS, both key signaling proteins in FID, were significantly reduced in coronary arterioles from old control and OVX rats. Dea‐NONO‐ate, an exogenous NO donor, produced similar vasodilation in all groups. These data indicate that endothelium‐dependent FID is impaired with advancing age in coronary arterioles from female rats, and estrogen‐replacement can improve or restore FID in OVX rats. NIH: HL077224‐02.
Spatial Interaction of the L‐type Calcium Channel with α5β1 Integrin
Authors: Chao J‐T. S. , Gui P. , Zamponi G. W. , Davis G. E. , Davis M. J.
Published: June 2021

α5β1 integrins play essential roles in modulating neuronal and vascular smooth muscle cell function by altering Ca2+ entry through the L‐type calcium channel (CaL). We previously demonstrated that an increase in Ca2+ current following α5β1 integrin activation is mediated by PKA and Src phosphorylation of CaL. Here, the spatial interaction of CaL with α5β1 integrin was examined using immunofluorescence (IF) confocal microscopy and co‐immunoprecipitation (IP). HEK293 cells expressing neuronal CaL were plated on an extracellular matrix substrate prior to IF or IP. Co‐association of CaL with β1 integrin was only detectable by IP in cells plated on fibronectin (FN), not on poly‐l‐lysine. By IF, about 60% of wild type (WT) CaL co‐localized with β1 integrin on FN, using paxillin‐vinculin colocalization as a reference. Marginal decreases (~90% of WT) in the degree of CaL colocalization with β1 integrin were seen for CaL constructs missing the proline‐rich domain (PRD) or the last 281 amino acid residues (Stop5). By IP, the degree of CaL co‐association with β1 integrin on FN was decreased to 59% and 79% of WT in the PRD and the Stop5 mutants, respectively. Significantly less co‐association (12% of WT) of CaL with β1 integrin was observed in a CaL construct with altered phosphorylation sites for both PKA and Src. In patch clamp studies, α5β1 integrin activation increased CaL current by 107% in WT, 10% in the Stop5 mutant, and by 88% in the PRD mutant. Overall, our data suggest that the co‐association of CaL with β1 integrin depends on the activation of β1 integrin by FN and subsequent phosphorylation of CaL by PKA
Role of adenosine A2a receptor in regulation of cerebral blood flow (CBF) during induced hypotension
Authors: Kusano Yoshikazu , Miekisiak Grzegorz , Kulik Tobias , Sandler Adam , Winn Richard H.
Published: June 2021

Introduction:

The aim of this study is to evaluate the role for adenosine A2a receptor during induced hypotension in mice.

Methods:

Anesthetized and ventilated wild type (WT) and adenosine A2a receptor knockout (KO) mice (C57Bl/6) were used. Catheters were placed in femoral arteries for measurement of mean arterial pressure (MABP) and withdraw the arterial blood. MABP was lowered to 35 mmHg by continuously withdrawing arterial blood at a rate of 25 μl/min. CBF was measured using the laser‐Doppler flowmetery through the intact calvarium. We also studied the effects of specific A2a receptor antagonist, ZM‐241385 (1 mg/Kg and 5 mg/Kg, ip).

Results:

During hypotension CBF was attenuated by 38.9% in WT (n=5) and 51.1% in KO mice (n=7) (P<0.05). The low dose of ZM‐241385 (1 mg/Kg) (n=6) had no effect on reduction of CBF. However with a higher dose (5 mg/Kg) (n=6), the CBF significantly reduced by 51.3% (P<0.05).

Conclusions:

These results suggest that A2a receptor is involved in the attenuation of CBF during induced hypotension.

Supported by NIH‐NS 21076 (HRW)
Perfusate composition is a determinant of the hydrodynamically relevant endothelial glycocalyx thickness in vivo.
Authors: Potter Daniel Raymond , Jiang John , Damiano Edward
Published: June 2021

The endothelial‐cell glycocalyx surface layer is a highly hydrated structure that is constantly bathed in the perfusing fluid, typically blood in vivo. It is proposed that there is a constant exchange of molecules between the blood and the endothelial glycocalyx that is essential to the integrity of the layer. To test the effect of different prefusate compositions on the endothelial glycocalyx in vivo, a micromanipulator is used to introduce a micropipette into the lumen of one of the primary feed arterioles in a mouse cremaster‐muscle preparation. The micropipette then steadily perfuses the microvascular network fed by that arteriole with either native plasma, cell‐culture media containing 20% serum, or saline. Micro‐particle image velocimetry data is then collected from downstream microvessels. Velocity profiles extracted from the μ‐PIV data are then used to estimate the effective hydrodynamically relevant thickness of the glycocalyx. Preliminary data indicate that vessels perfused with saline have no detectable hydrodynamically relevant layer. Supported by NIH grant R01 HL76499.
Cx40 modulates agonist‐mediated vasoconstriction during lipopolysaccharide induced inflammation
Authors: Ouellette Yves , Sieck Gary C.
Published: June 2021

The role of gap junctional intercellular communication during the inflammatory process is not well understood. In particular, the role of vascular connexins (gap junction proteins) in regulating blood flow in response to inflammatory agents such as lipopolysaccharide (LPS) has not been fully investigated. Our hypothesis is that agonist‐mediated vasoconstriction is dependent on the presence of connexin 40 (Cx40) within the microvessel wall. Wild‐type (WT) and Cx40−/− animals were treated with LPS (15 mg/kg ip) or saline for 18 hours. Small mesenteric resistance arteries (190–220 μm) were isolated, mounted on glass cannulae, pressurized at 60 mmHg with no lumen flow and placed on the stage of an inverted microscope. External arteriolar diameter was measured and concentration‐response curves to phenylephrine (PE, 10−9−10−4 M) and to extracellular Ca2+ (0 to 2 mM) under depolarizing conditions (120 mM K+) were conducted. The half‐maximal effect (EC50) was determined for each dose response curve. LPS treatment resulted in hyporesponsiveness to PE in WT mice (EC50 2.07±0.45 mM vs 0.56±0.15 mM in shams) but resulted in hyperresponsiveness in Cx40−/‐ (EC50 0.27±0.03 mM vs 3.15±1.50 mM in shams). However, LPS treatment resulted in hyporesponsiveness to Ca2+ in both WT mice (EC50 0.39±0.03 mM vs 0.23±0.01mM in shams) and Cx40−/− (EC50 0.75±0.24 mM vs 0.23±0.03 mM in shams). We conclude that agonist‐mediated but not Ca2+‐mediated vasoconstriction is modulated by the presence of Cx40.
Identification of Vascular Gap Junction Protein Expression in the Cochlear Microvessels
Authors: Shi Xiaorui , Yamamoto Hiroshi , Nuttall Alfred L.
Published: June 2021

Using the immunohistochemistry combined with confocal microscopy technique and western blot analysis, we found that pericytes on the vessels of the spiral ligament have Connexin 40 (Cx 40) and Connexin 37 (Cx 37) in both guinea pigs and mice. In particular, Cx 40 was abundantly expressedin both endothelial cells and pericytes of the spiral ligament vessels. Also Cx 40 was intensively expressed in the intermediate‐like cells of the stria vascualris in the guinea pigs but not obviously in the mouse stria vascularis. There was no Cx 37 and Cx 40 signal in the endothelial cells and the pericytes of the stria vascularis vasculature. Also no detectable Connexin (Cx 43) was found in both the vessels of the spiral ligament and the stria vascularis. Our results are different from studies that assessed Connexin expression in the retinal or brain pericytes which do expressCx 43. The variation of Connexin expression in the different vasculatures may have important consequences for the difference of coordination and propagation of vascular signals and responses. The regional and animal species difference in expression heterogeneity of Cx 40 & Cx 37 indicates a possible functional and structural heterogeneity of pericytes in the vessels of the spiral ligament and the stria vascualris. Supported by NIH NIDCD R03 DC008888‐01A1 & NIH NIDCD DC 00105, DC 005983.
Mastergene leads for an arteriogenesis switch and self‐tolerance of epigenetic vascular remodeling variations: Monocyte‐derived non‐coding small hairpin shRNA bioaptamers [angiotropin‐related RNP ribokines]
Authors: Wissler Josef H.
Published: June 2021

AIM: Switching on arteriogenenesis and tolerance of epigenetic [non‐Mendelian] neovascular remodeling variations [e.g. angiogenesis, restenosis following percutaneous transluminal coronary angioplasty] by monocytes was investigated by morphogenesis assays and entitled purified endogenous monokines [Wissler et al., Protides Biol. Fluids 34: 517–536, 1986; Materialwiss. Werkstofftech. 32: 984–1008, 2001; Ann. N. Y. Acad. Sci. 961: 292–297, 2002; 1022: 163–184, 2004; Mol. Biol. Cell Suppl. 18: 124, 2007; FASEB J. 21: 519.5, 2007]. RESULTS: Extracellular monocytic edited, modified, redox‐ and metalloregulated shRNA [<200 bases] were found, sequenced and attributed to angiogenic switch mastergenes RPL27A/Rpl27a [genes encoding ribosomal protein L27A]. By metal ion‐structured 5‘CUG3‘‐hairpin loops, they may address homologous domains shared in proteofactors related to arteriogenesis and [epi]genetic information indexing of vascular epigenome, growth, enzymes, vasoregulation and metabolic syndrome, termed K/R3H [K/RxxxH]; i.e. ‐t/s/xK/R/q/n/hxxxH/y/n/q/e/d/r/kx7–9h/xx7–9h/xx5–20K/R/q/n/e/h‐ with accessory basic [R/K]n, R/K‐zipper, SR/K/RS and/or HxxxH/y/n/q segments and relationship to other canonical domains [HxxxH, ATCUN, CxxxC, fingers, L‐zipper, Rossmann‐fold]. CONCLUSIONS: Such RNA may switch on arteriogenesis in [endothelial] cells: They are earliest outputs in transcription [~98%], translation [~2%] and in estimated recognition repertoires, superior in diversity / specificity [>~1017] to immune proteins [~1013]. For tolerance, all attributes make them efficient and competent to recognize, address and integrate information flow on all possible molecular shapes back to genomic mechanisms.
Role of Bilirubin Oxidation Products in delayed Cerebral Vasospasm after Subarachnoid Hemorrhage‐ an in vitro study
Authors: Nair Sunil G , Clark Joseph F , Wurster William L , Pyne‐Geithman Gail J
Published: June 2021

Delayed Cerebral Vasospasm (CV) is a crucial and often fatal complication of Subarachnoid hemorrhage (SAH). The etiology and molecular mechanism of CV is still a mystery. Recent reports have shown that Bilirubin Oxidation Products (BOXes) are present in significantly greater concentrations in the cerebrospinal fluid of patients with SAH who suffer from vasospasm (CSFV) than in those who do not (CSFC). Previous studies have shown that BOXes are vasoactive in vivo and potentiate smooth muscle contraction similar to PKC activators and phosphatase inhibitors in vitro.

Using tissue bath apparatus, we examined the potentiation of phenylephrine (PE)‐induced force‐generation by various concentrations of BOXes in porcine carotid artery tissue rings. 1.5 – 7 μmol/L BOXes showed significantly increased potentiation of PE‐induced contraction when compared to vehicle. This was also observed with CSFV. We also studied the cytoplasm‐to‐membrane translocation of PKC's α and δ, and Rho in the presence of three different patho‐physiologically relevant concentrations of BOXes (0.2, 2 & 20μmol/L) at two‐points (20 minutes and 3 hours). Translocation of these proteins indicates their activation. Both PKC's and Rho were translocated in a time‐ and dose‐dependent manner by BOXes.

Therefore, we postulate that BOXes may be one of the major molecules responsible for the etiology of CV after SAH.
An antioxidant flavonol improves endothelial function in type 1 diabetic rats
Authors: Woodman Owen L , Malakul Wachirawadee
Published: June 2021

Diabetes increases oxidant stress and impairs endothelium‐dependent relaxation. We investigated whether the antioxidant 3′,4′‐dihydroxyflavonol (DiOHF) reduces superoxide (O2−) and preserves endothelial function in aortae from diabetic rats. In Sprague‐Dawley rats 8 weeks after streptozotocin (55 mg/kg iv) blood glucose was elevated (39.4±0.4 mmol/l) compared to citrate treated control (5.5±0.1 mmol/l, p<0.05) and, using lucigenin‐enhanced chemiluminescence, there was an increased aortic generation of O2− (control 670±101, diabetic 1535±249 units/mg dry weight, p<0.05). In aortic rings precontracted with U46619 ACh‐induced relaxation was impaired (Rmax control 79±2, diabetic 66±3%, p<0.01) whereas endothelium‐independent relaxation to SNP was unaffected. In the presence of DiOHF (10−4 M) there was a significant reduction in O2− generation (165±21 units/mg, p<0.01) and enhanced relaxation to ACh (Rmax 83±4%). 2 separate groups of rats (control and diabetic) were treated daily with DiOHF (5 mg/kg ip) for 7 days. DiOHF did not affect blood glucose (control + DiOHF 5.5±0.1 mmol/l, diabetic + DiOHF 38.8±0.5 mmol/l) but reduced superoxide generation in diabetic aortae (580±115 units/mg, p<0.05) and enhanced ACh‐induced relaxation (Rmax 80±4%, p<0.05). DiOHF, acutely or after 1 week treatment, reduces oxidant stress and preserves endothelium‐dependent relaxation in aortae from diabetic rats.
Hyperlipidemia Enhances Leukocyte Rolling in vivo by Involving Myd88‐Dependent Innate Immune Pathways
Authors: Pistea Adrian , Olofsson Katarina , Lindholm Marie , Zetterqvist Anna V , Öhman Jenny Nilsson , Gomez Maria F , Nilsson Jan , Thorlacius Henrik , Björkbacka Harry
Published: June 2021

Background and aims:

Absence of MyD88, a central signaling protein in innate immune defense, protects Apoe−/−mice from atherosclerosis (Björkbacka, Nat Med 2004). We aimed to investigate the involvement of MyD88 in the regulation of leukocyte‐endothelium interactions – a critical step in the formation of atherosclerotic lesions.

Methods:

Apoe−/−Myd88+/+ and Apoe−/−Myd88−/− mice were fed ad libitum chow or a high fat diet (HFD) for 4 weeks. Leukocyte rolling and adhesion were assessed in vivo in the femoral artery and vein by means of intravital microscopy; adhesion molecule expression was assessed by flow cytometry on leukocytes and by confocal microscopy in cerebral arteries.

Results:

Caloric intake was not affected by diet and no significant weight gain was noticed. HFD induced a significant increase in the number of circulating neutrophils in the Apoe−/−Myd88+/+ mice. HFD also increased the number of activated leukocytes, as indicated by increased CD11b surface expression. Leukocyte rolling was significantly increased by HFD in femoral veins of Apoe−/−Myd88+/+ mice, as compared to chow fed littermates or Apoe−/−Myd88−/− mice receiving HFD (p<0.01).

Conclusions:

High fat diet‐induced leukocyte rolling is MyD88‐dependent and may contribute to the leukocyte accumulation in the vascular wall observed in atherosclerosis.
The Sterile Gene Collection: Filling the Gap in the Functional Genomic Analysis of Cell Biological Processes in C. elegans
Authors: Oegema Karen , Green Rebecca , Audhya Anjon
Published: June 2021

The C. elegans embryo is an important system for the systematic characterization of genes required for cell growth, division and polarity. The main advantage of C. elegans is the structure of its germline, which allows the use of a standard RNAi‐based protocol to generate oocytes reproducibly depleted of targeted essential proteins via a mechanism that does not require intrinsic protein turnover. Capitalizing on this feature, the first two divisions of embryos in which the ~2000 C. elegans genes required for embryo viability were individually inhibited was screened using high information content differential interference contrast microscopy. This approach identified 660 genes whose inhibition results in classifiable defects. However, genes required for processes such as membrane trafficking and cortical remodeling were conspicuously absent. We reasoned that a prior role for such gene products during oogenesis precluded their analysis in embryos and that the collection of ~600 genes whose inhibition results in sterile worms represents a substantial untapped resource. To characterize this collection, we performed an RNAi screen monitoring gonad structure and oocyte production in a strain co‐expressing a GFP‐fusion protein that labels the plasma membrane and an RFP‐histone fusion that labels the chromosomes. In addition to ~15 new phenotypic classes, this screen has identified ~90 gene products required for membrane trafficking (~20 of which are novel), and a separate set of ~100 gene products whose inhibition gives a multi‐nucleate phenotype suggesting a cytoskeleton‐based role in cortical remodeling (~30 of which are novel). This screen complements previous characterizations of the embryonic lethal collection to provide systematic information on the entire collection of C. elegans genes important for subcellular processes.
5‐HT2A receptor mediated contractility of Ovine pulmonary arteries: Effects of maturation and chronic hypoxia
Authors: Goyal Ravi , Loftin Matthew , Derrick Allison , Nguyen Dan , Pearce William J , Longo Lawrence D , Wilson Sean M
Published: June 2021

Serotonin (5‐HT) signaling plays important roles in pulmonary vascular contractility and development. Several studies illustrate that 5‐HT mediated pulmonary arterial (PA) contractility adapts with development in sheep and rat and in response to chronic hypoxic (CH) stress in the rat. Our studies show that 5‐HT induced Ca2+ elevations in fewer PA myocytes from fetal relative to adult sheep suggesting there may be increases in receptor expression associated with maturation. Using wire myography approaches, the present study tested the hypothesis that CH and maturation increase the potency as well as efficacy of 5‐HT‐mediated PA contractility in sheep. The results show the EC50 to 5‐HT was unchanged with maturation in PA from normoxic sheep. However, the potency to 5‐HT was reduced with CH in fetus and increased in adult. The tension developed by 10 μM 5‐HT increased with maturation but not CH and no differences were observed when normalized to the tension due to 125 mM KCl. Moreover, in all groups the contractility to 1 μM 5‐HT was completely abolished by 100 nM Ketanserin, a selective 5‐HT2A antagonist. In conclusion, based on this and our previous studies the changes in 5‐HT efficacy and potency with maturation and CH are most likely due to altered receptor number, second messenger coupling or phenotypic alterations in PA myocyte function. (Support from NIH, Sigma Xi, and UM)
uPA Contributes To Impairment Of SNP and PGE2 Cerebrovasodilation After Brain Injury Through Activation of LRP and ERK MAPK
Authors: Armstead William M , Christine Amy J , Higazi Abd Al‐Roof , Bdeir Khalil , Cines Douglas B
Published: June 2021

Introduction: Pial artery dilation to prostaglandin (PG)E2 and the nitric oxide (NO) releaser sodium nitroprusside (SNP) are blunted but responses to papaverine unchanged after fluid percussion brain injury (FPI). Urokinase plasminogen activator (uPA) and ERK mitogen activated protein kinase (MAPK) are upregulated and contribute to impaired cerebrohemodynamics after FPI in the piglet. PA vascular activity is mediated by the low density lipoprotein receptor (LRP). This study investigated the role of uPA, LRP, and ERK MAPK in impaired cerebrovasodilation to PGE2 and SNP after FPI.

Methods: Lateral FPI (2 atm) was induced in anesthetized 1‐5 day old piglets equipped with a closed cranial window. CSF ERK MAPK was quantified by ELISA.

Results: Pretreatment with the antagonist of the vascular action of uPA, suPAR, blunted impairment of SNP and PGE2 dilation after FPI while responses to papaverine were unchanged. Similarly, pretreatment with the LRP antagonist RAP, the monoclonal antibody against LRP (Mab ag LRP) and the ERK MAPK antagonist, U 0126, all blunted impairment of SNP and PGE2 dilation post insult. In contrast, control IgG had no effect on FPI induced impairment of SNP and PGE2. Phosphorylation of CSF ERK MAPK was upregulated after FPI but such upregulation was blunted in animals pretreated with suPAR, RAP, and MAb ag LRP. Control IgG had no effect on FPI induced ERK MAPK upregulation. The ERK MAPK antagonist U 0126 blocked the upregulation of CSF ERK MAPK after FPI.

Discussion: These data indicate that uPA contributes to impairment of SNP and PGE2 cerebrovasodilation after brain injury through activation of LRP and ERK MAPK.
Differential regulation of Protein Kinase G activity by chronic hypoxia and maturation in fetal and adult ovine cerebral arteries
Authors: Williams James M , Abrassart Jenna , Pearce William J
Published: June 2021

In ovine cerebral arteries, cGMP‐mediated relaxation is differentially and potently influenced by both postnatal maturation and chronic hypoxia. Here we examine the hypothesis that these effects are mediated by changes in the enzymatic activity of Protein Kinase G (PKG). In 100,000XG artery homogenates, rates of phosphotransfer from ATP[γ32P] to a specific PKG substrate (BPDEtide) following PKG activation with 5 μM 8pCPT‐cGMP were significantly enhanced by hypoxia but depressed by maturation and averaged 2704 ± 304 (normoxic adult), 2438 ± 186 (hypoxic adult), 3129±225 (normoxic fetus) and 2663 ± 186 (hypoxic fetus) pmol ATP/mg protein/min. Total PKG abundances (via Westerns) were markedly greater in fetal than adult arteries under both normoxic and hypoxic conditions. PKGα abundance was similar in both groups, but PKGβ abundance was significantly enhanced in fetal compared to adult arteries. Apparent specific enzyme activity (phosphorylation rate normalized to PKG abundance) was greater in normoxic animals whereas postnatal maturation enhanced activity only in hypoxic animals. These differences suggest that during maturation and hypoxic acclimatization PKG activity is influenced mainly by changes in the abundance of the PKGβ isoform but that opposing changes in PKG specific activity tend to stabilize overall PKG activity as well as vasoreactivity to changes in cGMP. Supported by NIH HD31226 and HL64867
The cerebral metabolic ratio during exercise in patients with cirrhosis
Authors: Gam Christiane , Rasmussen Peter , Seifert Thomas , Secher Niels H , Larsen Fin S , Nielsen Henning Bay
Published: June 2021

Intense exercise decreases the cerebral metabolic ratio of O2 to carbohydrates (glucose + (1/2)lactate). It is not known whether these variables are influenced by exercise in patients with cirrhosis and seven cirrhotic patients (five treated with propranolol) underwent ergometer cycling to exhaustion. Arterial lactate increased to 5.0 ± 0.3 mM (mean ± SEM; P < 0.05) and arterial‐jugular venous (a‐v) difference from −0.01 ± 0.03 mM at rest to 0.30 ± 0.05 mM (P < 0.05). Arterial glucose was 7.0 ± 0.6 mM and its a‐v difference 0.64 ± 0.07 mM was not different from that at rest (0.54 ± 0.03 mM). In healthy subjects maximal exercise increased arterial lactate to 12.9 ± 1.2 mM, and the a‐v lactate and a‐v glucose difference were 1.46 ± 0.25 and 0.79 ± 0.10 mM, respectively (P < 0.05). While the cerebral metabolic ratio decreased from 5.5 ± 0.5 at rest to 3.8 ± 0.3 (P < 0.05) during exercise in healthy subjects when arterial lactate was ≈5 mM, the cerebral metabolic ratio was not changed exercise in patients with cirrhosis. During exercise in patients with cirrhosis, the cerebral metabolic ratio is not affected, as the cerebral extraction of glucose and lactate are lowered and fatigue does not relate to a reduction in the cerebral metabolic ratio. A beta1+2‐receptor antagonist may influence the cerebral substrate uptake during exercise.