bluuebunny/crossref_metadata_2025 huggingface dataset:
Unusual reactions to 5‐HT3 receptor antagonists in a child with rhabdomyosarcoma
Authors: Lee Candace Y.W. , Ratnapalan Savithiri , Thompson Margaret , Nathan Paul , Closs Jordan , French Amy , Punnett Angela , Garcia‐Bournissen Facundo , Ito Shinya , Koren Gideon
Published: June 2021

Ondansetron and granisetron are 5‐HT3 receptor antagonists commonly used for prevention of chemotherapy‐induced nausea and vomiting. Rare acute dystonic reactions to ondansetron have been reported previously. We report a case of abnormal facial movements associated with granisetron.

A boy with prostatic rhabdomyosarcoma and lung and bone metastases developed abnormal facial movements while receiving ondansetron 4 mg po q8h (5 mg/m2/dose). These movements consisted of involuntary blinking of the eyes, pursing of the lips, forward thrusting of the jaw and protrusion of the tongue. After excluding other potential causes, the diagnosis of ondansetron‐induced movement disorder was made. These movements decreased when the dose of ondansetron was reduced and resolved with its discontinuation. On admission for a subsequent cycle of chemotherapy, the patient was treated with granisetron 480 µg iv q12h (20 µg/kg/dose). On day 3, the patient developed repeated forward thrusting of the jaw and simultaneous blinking of the eyes, which were self‐limiting and less severe than those associated with ondansetron use. As vomiting was effectively prevented, granisetron was continued until completion of therapy. Health professionals need to be aware of these possible, albeit rare, reactions to ondansetron and granisetron in paediatric oncology patients.

Supported by Hospital for Sick Children Research Institute (CL, GK)
Covalent and Non‐covalent Interactions of Caveolin and Small Ubiquitin‐Related Modifier (SUMO)‐2/3
Authors: Fuhs Stephen Rush , Insel Paul A
Published: June 2021

Caveolins (Cavs), critical protein components of caveolae, scaffold numerous signaling proteins. Cavs‐1 and ‐3 have a SUMOylation consensus motif and tandem SUMO Interacting Motifs (SIMs). We hypothesized that: 1) Cav is covalently SUMOylated and 2) interacts non‐covalently with SUMO. In vitro SUMOylation assays, transfection of HEK cells and immunoblots of cardiac myocyte lysates revealed that SUMO‐1 and SUMO‐2/3 covalently modify cav‐3 in vitro while only SUMO‐2/3 conjugates occur in cells. Cav‐3 co‐immunoprecipitated with the SUMO E2 (Ubc9) and E3 (PIASy). Co‐transfection of PIASy enhanced SUMOylation of cav‐3 and mutation of the consensus site Lys (K38R) reduced SUMOylation by 50%. To investigate non‐covalent interactions, we incubated rat cardiac myocyte lysates with agarose‐bound SUMO‐1 or SUMO‐2/3 and found a preference for SUMO‐2/3. Our findings implicate SUMOylation as a posttranslational modification that regulates the function of caveolin. We speculate that non‐covalent interaction may be important for its covalent modification by providing selectivity for SUMO‐2/3 vs. SUMO‐1. (supported by NIH grants and PhRMA Foundation Predoctoral Fellowship).
Activation of ERK1/2 MAP kinase in rat liver in vivo after binge and chronic‐binge ethanol intake may augment liver injury
Authors: Aroor Annayya , Shukla Shivendra D
Published: June 2021

Liver injury caused by heavy alcohol consumption progresses from steatosis to steatohepatitis in some individuals. Binge ethanol intake is important factor in the progression to steatohepatitis in chronic alcoholics. Recent studies have implicated activation of extracellular regulated kinases (ERK1/2) to the actions of ethanol in cultured hepatocytes. In the present study, role of of ERK1/2 was examined in a clinically relevant animal model of progressive liver injury. This involved chronic ethanol administration for 4 weeks followed by a binge dose of ethanol (chronic‐binge) that culminated in an amplified liver injury. Eight week old Sprague Dawley rats were fed ethanol in liquid diet for 4 weeks followed by single binge dose of ethanol (5 gm/kg body weight) through intragastric administration for 4 hrs. Control rats were pair‐fed isocaloric diet. In the control for the binge ethanol was replaced by water. Chronic ethanol treatment (4 weeks) resulted in mild steatosis and necrosis whereas chronic‐ binge group exhibited marked steatosis and significant increase (2 fold) in necrosis. Chronic ‐ binge group also showed significant increase (2 to 3 fold) in the phospho‐ERK1/2 in liver with negligible effect on ERK1/2 protein level. Activation of ERK1/2 was not significant in the liver of ethanol alone treated group compared to control group. Taken together, these results suggest that activation of ERK1/2 by binge ethanol may augment steatosis and necrosis in liver. Supported by NIH grant AA 11962.
Phosphodiesterase 7 (PDE7) and PDE4/7 inhibitors kill chronic lymphocytic leukemia (CLL) cells via a cAMP‐mitochondrial‐dependent pathway
Authors: Zahno Anja Christina , Murray Fiona , Zhang Lingzhi , Rassenti Laura , Cottam Howard , Kipps Thomas , Insel Paul
Published: June 2021

Chronic lymphocytic leukemia (CLL), the major form of adult leukemia, is characterized by accumulation of B‐cells. We have found drugs that raise cAMP can kill malignant lymphocytes and (compared to normal peripheral blood mononuclear cells (PBMC) and normal B‐cells) CLL cells overexpress PDE7B, which selectively catalyzes the hydrolysis of cAMP. Furthermore, we found that CLL cells are more sensitive to PDE7 and PDE4/7 inhibitor‐induced apoptosis compared to normal B‐cells. Using CLL cells we sought to investigate the mechanism of PDE7 and PDE4/7‐induced apoptosis. Inhibition of PDE7 (30 μ]M BRL‐50481, 10 μM IR‐202) and dual inhibition of PDE4 and PDE7 (100 nM IR‐284) increased basal and forskolin‐stimulated cAMP accumulation in CLL cells, suggesting that their pro‐apoptotic effect occurs via a cAMP‐dependent mechanism. BRL‐50481, IR‐202 and IR‐284 all induced mitochondrial depolarization, assessed using a MitoProbe JC‐1 assay kit, (13 ± 4 %; 17 ± 6 %; 39 ± 5 % depolarization, respectively vs. vehicle) and cytochrome c release from the mitochondria into the cytosol, visualized by immunofluoresence. We conclude that PDE inhibitor‐induced apoptosis of CLL cells occurs via a cAMP‐mitochondrial‐dependent mechanism and that PDE7 inhibition may be a novel therapeutic approach in CLL. Supported by the Lymphoma and Leukemia Society, NIH and Swiss Cancer League.
Hexahydrocannabinols, novel synthetic cannabinoid derivatives, suppress the tumor growth by inhibiting the VEGF secretion and angiogenesis
Authors: Thapa Dinesh , Lee Jong Suk , Xia Likai , Lee Yong Rok , Kim Jung‐Ae
Published: June 2021

Cannabinoids, the active components of Cannabis sativa L. have been shown to exert antiproliferative actions in wide spectrum of tumor cells. However, the specific mechanism and target for these processes remain poorly understood. In the present study we examined anticancer potentials of hexahydrocannabinols. The synthetic hexahydrocannabinols inhibited the vascular endothelial growth factor (VEGF)‐induced angiogenesis on in vivo chick chorioalantoic membrane (CAM) assay. In addition, hexahydrocannabinols suppressed VEGF‐stimulated proliferation, tube formation, and migration in a concentration‐dependent manner. In MCF‐7 cells, hexahydrocannabinols concentration‐dependently inhibited VEGF secretion. Furthermore, we found that the hexahydrocannabinols dose‐dependently inhibited tumor growth and tumor‐induced angiogenesis. In addition, these synthetic cannabinoids significantly inhibited the TPA‐induced NF‐κB activation, which is known to regulate many important genes for inflammation, angiogenesis, and tumor growth. Overall, these findings strongly suggest that synthetic hexahydrocannabinols may be the promising compounds for antitumor drug development.
Intestinal microvascular changes associated with the induction and perpetuation of chronic gut inflammation
Authors: Harris Norman R. , Whatley Joseph R. , Carter Patsy R. , Morgan Georgia A. , Grisham Matthew B.
Published: June 2021

Adoptive transfer of naïve CD4+ T‐cells into lymphopenic mice induces chronic small and large bowel inflammation similar to Crohn's disease. Although much is now known regarding the immunopathology in this model of inflammatory bowel disease, virtually nothing is known about the microvascular hemodynamic changes in the induction and perpetuation of chronic gut inflammation. In this study, CD4+CD45RBhigh T‐cells obtained from healthy C57BL6 donors were transferred into lymphopenic recombinase activating gene‐1 deficient (RAG ko) mice, and in the following weeks, intravital microscopy was used to examine the ileum and proximal colon. Measurements included arteriolar diameters and the velocity of labeled platelets (indicator of blood velocity), with the results compared to unreconstituted RAG ko controls. In < 1 wk following reconstitution, velocity and wall shear rate of the arterioles decreased by > 50% compared to controls, with this decrease also observed at 4‐5 and 7‐9 weeks post‐reconstitution. At 7‐9 weeks, arteriolar diameters were found to be ~15% larger than in controls, but despite this dilation, flow rates in the individual vessels were decreased by ~30%. Whether the overall intestinal flow rate is decreased in this model may be dependent on the reported angiogenesis, which could explain the lower individual vessel flows despite the late vasodilation. Supported by Projects 1 & 2 of NIH P01DK043785.
Platelets enhance neutrophil transendothelial migration
Authors: Lam Fong , Burns Alan , Smith C. Wayne , Rumbaut Rolando
Published: June 2021

Platelets are increasingly recognized as important mediators of inflammation in addition to thrombosis. While platelets have been shown to promote neutrophil (PMN) adhesion to endothelium in various inflammatory models, it is unclear whether platelets enhance neutrophil transmigration across inflamed endothelium. To test this hypothesis, we studied the effects of platelets on PMN transendothelial migration both in vitro and in vivo, using interleukin‐1 β‐stimulated human umbilical vein endothelial cells (HUVEC), and a well‐characterized corneal injury model in C57BL/6 mice. In the HUVEC model, PMN in the presence of platelets demonstrated enhanced transendothelial migration under stationary conditions (% transmigration at 500 sec was 57±6% vs. 33±5%; p<0.01). Further, in a parallel‐plate chamber under physiological flow conditions, platelets increased the speed of PMN transmigration following firm adhesion (duration of transmigration was 34.4±2.7 vs. 51.4±2.9 sec; p<0.01). In vivo, platelet depletion reduced neutrophil emigration from limbal vessels 60 minutes after injury (280±12 vs. 513±44 PMN; p<0.05). These data support the notion that platelets enhance neutrophil migration across inflamed endothelium both in vitro and in vivo. The mechanisms involved remain to be elucidated.

Support: T32 HL07747‐15; RO1 EY018239
The attenuation of leukocyte‐endothelial interactions by a unique broad‐spectrum protein kinase C inhibitor (Gö 6983) in rat mesenteric postcapillary venules
Authors: Chen Qian , Rueter Brian , Young Lindon Howard
Published: June 2021

Protein kinase C (PKC) is a very important enzyme that regulates endothelial and leukocyte function. However, the role of broad‐spectrum PKC inhibition involving the atypical zeta isoform in regulating leukocyte‐endothelial interactions has not been clarified in vivo in real‐time. The microcirculation of postcapillary venules in rat mesentery was observed by intravital microscopy. We found that superfusion of NG‐nitro‐L‐arginine methyl ester (L‐NAME, a nitric oxide synthase (NOS) inhibitor, 50 µM, n=10) significantly increased leukocyte rolling, adherence and transmigration within a 2 hour period compared to superfusion of Krebs' buffer (n=8) (P<0.01). By contrast, in the presence of Gö 6983 (25 nM, n=5/ 100 nM, n=5/ 200 nM, n=6), a unique broad‐spectrum inhibitor of PKC that inhibits isoforms from all three biochemical classes, these leukocyte‐endothelial interactions were dose‐dependently attenuated compared to L‐NAME alone (P<0.05). Moreover, Gö 6983 dose‐dependently decreased superoxide release from neutrophils compared to control (P<0.01). In summary, inhibition of endothelial NOS activity (i.e., superfusion of L‐NAME) enhances leukocyte‐endothelial interactions. By contrast, inhibition of PKC can decrease these increased interactions, which may be related to decreasing superoxide release from neutrophils. This study was supported by Center for Chronic Disorders of Aging at PCOM.
Protein kinase C beta II inhibitor attenuates leukocyte‐endothelial interactions during acute endothelial dysfunction
Authors: Rueter Brian Matthew , Chen Qian , Young Lindon Howard
Published: June 2021

Vascular dysfunction in diabetic patients and animal models is characterized by an increased inflammatory response that is mediated by increased leukocyte‐endothelial interactions. Protein kinase C (PKC) inhibition has been attributed to attenuate these interactions. However, the role of a selective PKC beta II inhibitor regulating endothelial‐leukocyte interactions has not been evaluated in vivo in real‐time. The effect of PKC beta II peptide inhibitor on leukocyte‐endothelial interactions in rat mesenteric postcapillary venule circulation was determined by using intravital microscopy. We found that superfusion of NG‐nitro‐L‐arginine methyl ester (L‐NAME, 50 µM), a nitric oxide synthase inhibitor (n=10), significantly increased leukocyte rolling, adherence, and transmigration within a 2 hour period compared to Krebs' buffer control rats (n=7, P<0.01). However, PKC beta II inhibitor (1.0 µM, n=8/ 5.0 µM, n=7) dose‐dependently attenuated these leukocyte‐endothelial interactions compared to L‐NAME alone (P<0.05). Moreover, PKC beta II peptide inhibitor dose‐dependently decreased superoxide release from neutrophils compared to controls (P<0.01). In summary, the PKC beta II inhibitor attenuates leukocyte‐endothelial interactions that may be related to inhibiting superoxide release from neutrophils. This study was supported by Center for Chronic Disorders of Aging at PCOM.
Protective effect of 11,12‐epoxyeicosatrienoic acid in lung ischemia‐reperfusion injury
Authors: Townsley Mary , Barnes Sue , Morisseau Christophe , Hammock Bruce , King Judy
Published: June 2021

Epoxyeicosatrienoic acids (EETs) are protective in myocardial and brain ischemia, an effect variously attributed to activation of KATP channels or blockade of adhesion molecule expression. This study addressed whether EETs would attenuate the increase in permeability induced by 45 min ischemia‐30 min reperfusion (IR) in isolated, buffer‐perfused rat lungs. The filtration coefficient (Kf), a sensitive index of endothelial permeability, increased 10.5±2.5‐fold (mean±SE) after IR alone (p<0.05), but not in time controls. Blockade of endogenous EET degradation with dicyclohexylurea (50 ?M) or compound 950 (100 ?M) did not significantly attenuate the response to IR (Kf increased 5.5±0.9‐fold). In lungs treated with 11,12‐EET (300 nM) at reperfusion, Kf only increased 2.5±0.6‐fold (p<0.05 vs IR). 14,15‐EET (300 nM) administered at reperfusion had no significant benefit. The beneficial effect of 11,12‐EET was not prevented by blockade of KATP channels (glibenclamide, 10 μM) nor by blockade of TRPV4 (ruthenium red, 3 μM). Finally, 11,12‐EET‐dependent alteration in adhesion molecules expression is unlikely to explain its beneficial effect, since the expression of the adhesion molecules VCAM and ICAM in lung after IR was similar to that in time controls. We conclude that 11,12‐EET has benefit in the setting of lung IR, though understanding its mechanism will require further study. Supported by HL067461.
Effect of pressure overload on cardioprotection of postconditioning: comparison to pressure unloading
Authors: Mozaffari Mahmood , Liu Jun
Published: June 2021

This study tests the hypothesis that pressure overload attenuates cardioprotective effect of postconditioning. Accordingly, following stabilization, Langendorff‐perfused hearts of male rats were subjected to regional ischemia (I; 40 min) followed by reperfusion (R; 2 hrs) with perfusion pressure set at either 80 or 160 cmH2O. Postconditioning was induced by six cycles of I and R (10 sec. each) immediately at the end of index I followed by continuous R (n=7‐10 hearts/condition). Comparisons were made to pressure unloading which consisted of perfusion of heats at 160 cmH2O during the stabilization and I periods followed by switchover to 80 cmH2O during R (n=6 hearts). Elevation in perfusion pressure resulted in larger infarcts (p<0.05). Postconditioning reduced infarct size at either pressure (p<0.05); nonetheless, a significant differential persisted between hearts perfused at the high and low perfusion pressures. Interestingly, pressure unloading markedly reduced infarct size, an effect more prominent than that conferred by postconditioning. The results provide evidence for the deleterious impact of pressure overload on postconditioning‐induced cardioprotection. Further, the marked cardioprotective effect of pressure unloading suggests involvement of cardioprotective pathways that may be distinct from those mobilized by postconditioning. Supported by AHA, Southeast Affiliate.
A Murine Model of Acute Hindlimb Ischemia/Reperfusion Injury
Authors: TU HUIYIN , LI YULONG , TRAN PAUL
Published: June 2021

Introduction

15% of preventable combat fatalities are due to hemorrhage from extremity wounds, making emergency tourniquet one of the first‐line treatments. While lifesaving, tourniquets can cause serious ischemia‐reperfusion (IR) injury. Fundamental to IR injury is mitochondrial production of superoxide. We refined a murine model of acute hind limb IR to simulate the clinical use of tourniquet.

Methods

Groups of C57/BL6 mice underwent an IR protocol of three hours of ischemia and four hours of reperfusion. IR was achieved by placement and release of a rubber tourniquet around the hind limb, at the greater trochanter level. Muscle blood flow (MBF) was measured using a Doppler probe. Muscle necrosis was measured using TTC stain. Superoxide production was measured using chemiluminescence and DHE staining. MnSOD protein expression and activity were measured using Western blot and a modified nitrite method. Mitochondrial respiratory chain function was measured via spectrophotometry.

Results

Significant ischemia and microcirculatory dysfunction were achieved: MBF during IR was < 10% and < 41% of baseline, respectively (n=8), accompanied by >40% necrosis (n=9). Superoxide production was increased (0.05 vs 0.13, p<.05 sham vs IR)accompanied by concomitant increases in MnSOD activity and protein expression (1.51 vs 2.26 , p<.05,sham vs IR, n=8). Mitochondrial complexes I‐IV showed signs of dysfunction (Complex I‐ IV: 0.13, 0.31, 1.37, 2.76 sham vs 0.08, 0.21, 0.7, 1.97 IR, p<.05 ).

Conclusions

We have demonstrated an animal model of clinical tourniquet use. IR produces significant mitochondriopathy which is associated with elevated superoxide production and antioxidant activity. The model can be used to mechanistically evaluate antiischemic molecules in efforts to minimize the morbidity and mortality of battlefield injury.
CULTURE OF LYMPHATIC VESSELS AND DEVELOPMENT OF TRANSFECTION TECHNIQUES TO TARGET GENES INVOLVED IN REGULATION OF LYMPHATIC CONTRACTILITY
Authors: Gashev Anatoliy A. , Dougherty Patrick , Gasheva Olga Yu. , Nepiushchikh Zhanna V. , Wang Wei , Kelly Katherine A. , Cai S. , Periasamy M. , Babu G. J. , Meininger Cynthia J. , Davis Michael J. , Muthuchamy Mariappan , Zawieja David C.
Published: June 2021

The power and specificity of gene transfection has never been tested on lymphatic vessels (LV). We have optimized the organ culture technique to maintain the viability of isolated rat LV for 7‐9 days that would be sufficiently long to overexpress or knock down genes of interest. We used an adenovirus expressing GFP to test the transfection efficiency ‐ resulting in ~80‐90% GFP expression in lymphatic muscle cells with normal LV contraction patterns. We transfected several genes involved in the regulation of lymphatic contractility. After 72 hours of overexpression of SERCA2a in LV muscle cells, functional tests demonstrated diminished lymphatic tone (predicted lowered cytoplasmic Ca++) and ~50% higher lymphatic phasic amplitude (predicted enhanced systolic Ca++ release). We specifically knocked down of 80‐95% MLCK in LV ‐ both tone and phasic contractions were greatly diminished. Further refinement of the organ culture conditions allowed us to monitor LV up to 12 days under controlled pressure conditions. This experimental model allowed us to establish controlled, high‐pressure conditions (10 cm H2O) for 4‐5 days with first reversible signs of pump failure after 2‐3 days; the model can be used as an accessible and reasonable simulation of the lymphatic pump alterations during lymphedema.
Characterization of VIP‐induced contractile inhibition in lymphatic vessels
Authors: von der Weid Pierre‐Yves , Rehal Sonia , Dyrda Peter
Published: June 2021

Lymphatic vessels actively remove interstitial fluid through rhythmical, phasic contractions of the muscle layer in the vessel wall. This activity is critical during inflammation to prevent excessive edema. Importantly, lymphatic pumping is modulated by inflammatory mediators. In particular, it is inhibited by vasoactive intestinal peptide (VIP), a neuro‐ and immuno‐modulator with anti‐inflammatory properties, detected in lymphatic innervation and in immune cells patrolling the lymph. Using electrophysiology, pharmacology and molecular biology techniques we aimed at characterizing the mechanisms involved in the inhibitory effect of VIP in lymphatic vessels of the guinea pig mesentery. RT‐PCR analysis revealed expression of VPAC1, VPAC2 and PAC1 receptor mRNAs in lymphatics. VIP‐induced pumping inhibition was shown to be concentration‐dependent (EC50 15 nM) and accompanied by a hyperpolarization (range 4‐15 mV, n=4‐22). The PAC1 antagonist PACAP(6‐38) most effectively blocked VIP‐induced responses (n=4). VIP‐induced responses were not affected by nitric oxide synthase and cyclooxygenase inhibitions (n=4), but depend on protein kinase A and KATP channel activation (n=4‐9). This inhibitory action of VIP needs to be considered during inflammation since high VIP concentrations in the vicinity of lymphatics may compromise lymph drainage

Supported by the Crohn's and Colitis Foundation of Canada
Phosphodiesterase 4 and 5 are Dominant cAMP‐ and cGMP‐hydrolyzing Isoforms in Rat Skeletal Muscle Arteries and Venules
Authors: Wang Jianjie , Kazmi Murtaza , Huxley Virginia H.
Published: June 2021

Phosphodiesterases (PDEs) hydrolyze cAMP and cGMP, important second messengers controlling blood flow, vascular exchange, and remodeling. PDE inhibitors have been used clinically in many disease states including inflammation, asthma, septic shock, and ischemia. Our previous study showed the PDE expression profile in primary cultured microvascular endothelial cells (FASEB J. 2008 22:1145.4). To understand the basis of PDE in the regulation of vascular function in vivo, we first identified the distribution of PDE isoforms in intact arteries and venules of abdominal skeletal muscle from strain‐ and age‐ matched male and female rats. PDE mRNA was quantified via real‐time RT‐PCR assay. Five PDE isoforms (PDE1, PDE2, PDE3, PDE4, and PDE5) were expressed in all arteries and venules. The expression levels were: 1) cAMP‐hydrolyzing PDE, PDE3<PDE4; 2) cGMP‐hydrolyzing PDE, PDE1<PDE5; 3) both cAMP‐ and cGMP‐hydrolyzing PDE, PDE2<PDE4(cAMP‐hydrolyzing)< PDE5(cGMP‐hydrolyzing). Subisoforms of PDE1 (A, B, C), PDE2A, PDE3 (A, B), PDE4 (A, B, D), and PDE5A were found in rat skeletal muscle arteries and venules from all juvenile (<40 days) and adult (>3 months) males and females. The findings indicate PDE1‐5 are present in all vessels while PDE4 and PDE5 are dominant cAMP‐ and cGMP‐hydrolyzing PDE isoforms, respectively. (Supported by NIH RO1HL078816 and NASA NNJ05HF37G. *The first 2 authors contributed equally to this work).
Alignment‐dependent regulation of vangl2, a planar cell polarity core member, in adult endothelial cells
Authors: Rekapally Harish , Stiening Chad M , Hoying James B
Published: June 2021

Proper vessel inflow‐outflow polarity is important for vascular health. We hypothesized that the planar cell polarity (PCP) molecular program, which establishes polarity in embryonic tissue fields, participates in adult EC polarized behavior. We show, for the first time, that Vangl2, a core PCP member, is present in adult ECs, is expressed throughout neovascularization and is regulated by shear stress‐dependent EC re‐alignment. Under static conditions, Vangl2 is located in punctate structures within the cytoplasm of cultured human iliac endothelial cells (HIAEC). Upon exposure to 20 dynes/cm2 for 24 hrs, Vangl2 protein expression is upregulated and appears within the Golgi complex. Continued exposure of EC to shear stress without re‐alignment maintains elevated Vangl2 expression, but Vangl2 is lost from the Golgi complex. However, when cell re‐alignment is induced during continued flow exposure, Vangl2 re‐appears in the Golgi and its expression is increased further suggesting that cell alignment and not shear stress, per se, is the stimulus for Vangl2 regulation. These findings demonstrate the active presence of the PCP program in adult endothelial cells and, given its conserved role in organizing cell signaling, may act to coordinate EC activity associated with cell polarization and, therefore, may be important in the control of vascular maturation and organization. Supported by AHA 0755710Z.
Inhibition of eNOS internalization dissociates PAF‐induced eNOS phosphorylation at Ser1177 from NO production
Authors: Sánchez Fabiola Alejandra , Rana Roshniben , Kim David D , Iwahashi Toru , Durán Walter N
Published: June 2021

We demonstrated earlier that caveolar endocytosis of eNOS regulates PAF‐induced endothelial permeability (AJP Heart 295: H1642 ‐ H1648 2008). We tested further the hypotheses that caveolar traffic is a requirement for the development of a robust hyperpermeability response and that eNOS‐derived NO production is associated to caveolar translocation. To this end, we used ECVCD8eNOS‐GFP and CVEC (bovine coronary postcapillary venular endothelial cells), as a model and PAF as the agonist. We inhibited caveolar traffic using cyclodextrin, dynamin 2K44A (negative mutant of dynamin) and cav1‐Y14F (negative mutant of caveolin1 that prevents caveolin‐1 phosphorylation at Y14). Cyclodextrin and the dominant negative mutants inhibited eNOS translocation via caveolae. eNOS phosphorylation at Ser1177 was enhanced in CD8eNOS‐GFP and CVEC when cyclodextrin or dynamin 2K44A were applied, and the response was cell line dependent when cav1‐Y14F was applied , i.e., CD8eNOS‐GFP demonstrated increased phosphorylation at Ser1177, but CVEC did not. Interestingly, NO production was partially inhibited by cav1‐Y14F and completely inhibited by dynamin 2K44A and cyclodextrin. We obtained a direct correlation between PAF‐induced eNOS traffic via caveolae and hyperpermeability. Our results demonstrate for first time an uncoupling between eNOS phosphorylation and NO production. Indeed, our results suggest a mechanism where eNOS is activated by phosphorylation at the plasma membrane and endocytosis is required to cause hyperpermeability. (Supported by 5R01 HL070634 and 1RO1 HL088479).
Vascular endothelial growth factor (VEGF) receptors 1 and 2 gene expression is up‐regulated in the cervix of pregnant mice treated with lipopolysaccharide (LPS).
Authors: Thompson Morgan Paige , Mowa Chishimba Nathan
Published: June 2021

Cervical remodeling entails a physiological inflammatory response, which can be induced by inflammatory agents, such as lipoplysaccharide (LPS), and inflammation is closely associated with vascular remodeling. We have previously characterized trends in levels of vascular endothelial growth factor (VEGF) and some of its signaling molecules during cervical remodeling, and administration of LPS to pregnant mice has been shown to enhance VEGF synthesis in the cervix. Here, we investigate whether LPS‐induced inflammatory effects on VEGF also regulate levels of its receptors, KDR and Flt‐1, in a dose‐dependent and time‐dependent manner. LPS dissolved in normal saline was administered intra‐cervically to three groups of mice at day 12 of pregnancy; control, a 250ng dose, and a 300ng dose. The cervix was harvested 6 hours later and levels of VEGF receptor levels were analyzed by real‐time PCR. Also, LPS was administered intra‐vaginally to three groups of mice at day 12 of pregnancy and the cervix was harvested for control, 3 and 5 hours later, and analyzed by real‐time PCR. Dose and time‐dependent data reveals that the gene expressions for both VEGF receptors are up‐regulated by LPS. We conclude that LPS‐induced inflammation may be mediated, among other pathways, through VEGF‐VEGF receptor pathways.
Temperature controlled small animal platform for in vivo microcirculation studies
Authors: Scheider Linda , Berger Brian , Torres Filho Ivo
Published: June 2021

Mouse models are becoming more representative of human studies with the complete mapping of the mouse genome. Anesthetized mice may easily become hypothermic and exposed tissues are highly affected by environmental temperature changes. In certain applications, superfusion is not necessary: a dry, compact, inexpensive, temperature controlled platform with a clear viewing field is needed for these studies. We describe two designs for animal platforms with these characteristics. The platforms include a body heater and heated pedestal for tissue observation under an upright microscope. In the fixation area for the exposed tissue, a metal cylinder heats an isolated water column. The platforms use feedback sensors and controllers to maintain a constant body temperature. One platform is very compact and is about 13 mm in height for optimal distance between the substage condenser and the microcirculation. The second is slightly taller but uses all readily available inexpensive parts for easy assembly. These platforms are ideal for studies with transillumination of small animal microcirculation. Testing of the platforms showed constant temperatures were held with time and the components are durable and reliable.

Support

AD Williams Trust Funds, VCU.
Resonance Raman spectroscopy of human sickle cell hemoglobin from transgenic mice
Authors: Torres Filho Ivo P , Terner James , Scheider Linda
Published: June 2021

We have previously developed a methodology to measure hemoglobin (Hb) oxygen saturation using Raman spectroscopy in vivo (AJP 289:H488, 2005; JAP 104:1809, 2008). However, the technique has not yet been used in sickle cell disease (SCD). We have established a colony of transgenic (Tg) SCD mice. This strain no longer expresses mouse Hba and Hbb, but express human sickle hemoglobin (HbS). It mimics the genetic, hematologic and histopathologic features found in humans with SCD. Whole blood samples were obtained from anesthetized Tg mice and normal control mice. Raman spectra from these samples were obtained in vitro using the exciting wavelengths in the range of 405 ‐ 532 nm emitted by a tunable laser. The scattered light was sampled at 90 degrees using a 0.5 m spectrometer and a nitrogen cooled CCD detector. In order to induce HbS polymerization, Raman spectra were collected from blood samples exposed to high and low oxygen environments. The findings suggest that strong Raman signals can be obtained from normal and SCD blood, opening the possibility for in vivo noninvasive measurements of hemoglobin Hb and HbS saturation in the microcirculation. The resonance Raman technique should provide additional worthwhile microscopic information in the study of the abnormal hemoglobins in vivo.

Support

VCU.
Extensive Endothelial Damage in an Inflammatory Model of Pulmonary Arterial Hypertension
Authors: Mathew Rajamma , Huang Jing , Wolk John H , Gewitz Michael H
Published: June 2021

Pulmonary arterial hypertension (PAH) is a progressive disease associated with right heart failure and premature death. Diagnosis of PAH is often made late and available therapy fails to reverse the disease process. To test the hypothesis that the poor response to therapy in PAH is due to the involvement of multiple signaling pathways, we investigated time‐dependent changes in the expression of caveolin‐1, PY‐STAT3, Bcl‐xL, and the molecules involved in NO signaling pathway (eNOS, HSP90, Akt) in rats at 48 hr, 1, 2, and 3 wks post‐monocrotaline (MCT). Progressive PAH, right ventricular hypertrophy and pulmonary vascular remodeling were observed at 2 and 3 wks post‐MCT. Progressive loss of endothelial caveolin‐1 coupled with activation of PY‐STAT3 and upregulation of Bcl‐xL preceded the onset of PAH. There was an early and progressive loss of Tie2 which colocalizes with caveolin‐1 and has been implicated in PAH. Expression of eNOS increased at 48 hr and 1 wk post‐MCT with a significant loss at 3 wks post‐MCT. At 2 wks post‐MCT concomitant with PAH, the expression of eNOS activating molecules HSP90 and Akt was significantly reduced, which is consistent with impaired NO availability as observed previously. We conclude that there is progressive deregulation of multiple signaling pathways leading to cell proliferation, inhibition of apoptosis and NO impairment in PAH. This may in part, explain the difficulties encountered in the treatment of PAH.
Superoxide – NO interaction plays an important role in inhibition of hypoxic pulmonary hypertension by hypercapnia
Authors: Chovanec Milan , Hnilickova Olga , Smolkova Pavlina , Herget Jan
Published: June 2021

Superoxide, nitric oxide (NO) and peroxynitrite play a pathogenetic role in the development of hypoxic pulmonary hypertension (HPH).

Rats exposed to normobaric hypoxia with hypercapnia (10% O2+4.5% CO2, n=7) for 3 weeks had significantly lower (22.9 vs. 30.0 mmHg, p<0.01) PAP and right ventricle weight (RV/LV+S ratio, 34.4 vs. 54.0, p<0.0001) compared to rats exposed to hypoxia with no hypercapnia (10% O2, n=9).

We tested the hypothesis that superoxide ‐ NO interaction plays role in inhibition of HPH by hypercapnia. We measured the expired amount of NO of living rats and the level of NOx in plasma by the chemiluminescent method and the plasma concentration of nitrotyrosine (a marker of peroxynitrite) by ELISA on male adult Wistar rats exposed to normobaric hypoxia ‐ 10% O2 (group H, n=6) or normobaric hypoxia ‐ 10% O2 with hypercapnia ‐ 4.5% CO2 (group H+CO2, n=6) for 4 days. Group N (n=6) were normoxic controls. The amount of expired NO (689.3 vs. 889.2 pg.min−1, p<0.05), the level of NOx in plasma (57.4 vs. 97.2 µM, p<0.01) and the plasma concentration of nitrotyrosine (7.9 vs. 9.9 µM, p<0.05) were significantly decreased in group H+CO2 compared to group H.

Superoxide ‐ NO interaction plays an important role in inhibition of HPH by hypercapnia.

Supported by Cardiovascular Research Centre MSMT 1M 0510 and Grant: GAUK 7780/2007
Alcohol intoxication suppresses blood‐borne mediators which signal hematopoietic changes during pneumococcal pneumonia.
Authors: Raasch Caroline Elizabeth , Zhang Ping , Neslon Steve , Bagby Greg J
Published: June 2021

Alcoholic patients with pneumonia frequently present with low leukocyte counts, predictive of poorer outcomes. Our previous studies have shown alcohol intoxication attenuates the induction of Sca‐1 surface protein in lineage− c‐kit+ cells during infection, which is associated with granulocytopenia and increased mortality. To investigate how alcohol suppresses Sca‐1 induction during pneumonia, plasma collected from mice 24 h after i.p. saline or alcohol and i.t. S. pneumoniae was added to lineage− c‐kit+ Sca‐1− bone marrow cells isolated from naïve mice. After 24h in culture, cells were analyzed for Sca‐1 surface protein. Cells exposed to plasma from pneumonia infected animals demonstrated a 4.4‐fold increase in Sca‐1+ cells over control levels (p < 0.05), but no increases were seen in response to plasma from mice intoxicated at the time of infection. As Sca‐1 re‐expression contributes to the pneumonia‐induced increase in stem cell number, alcohol intoxication may suppress the hematopoietic response to pneumonia in part by impairing the release of a blood borne mediators. Supported by NIH AA07577 & AA09803.
Soluble CD40 ligand stimulates functional microparticle release from pulmonary microvascular endothelium
Authors: Rai Jyoti , King Judy A , Moore Tim M , Bauer Natalie N
Published: June 2021

Plasma sCD40L levels are elevated during diseases involving activation of immune, inflammatory, and coagulation cascades. During inflammatory states microparticles, small vesicular segments of cell membrane, increase yet it remains unknown whether the pulmonary vascular bed contributes to the increase in number and what the function of these microparticles may be. Our hypothesis is that sCD40L stimulates an increase in endothelial microparticle (EMP) release from the pulmonary microvasculature which function as regulators of vascular repair. Treatment of PMVECs with sCD40L (1 µg/mL, 1 hour) stimulated a two‐fold increase in EMP number as compared to basal EMP production. This increase was inhibited by pre‐treatment of PMVECs with anti‐CD40 antibody (0.5ug/ml, 1 hr.). When PMVEC monolayers were disrupted with trypsin, treatment of these monolayers with sCD40L‐EMPs significantly reduced barrier resealing. It has previously been reported that PMVECs express activated leukocyte cell adhesion molecule (ALCAM) and suggested that ALCAM plays a role in barrier integrity. To determine whether ALCAM was expressed in EMPs we examined them by TEM. We determined that sCD40L‐EMPs carry ALCAM which may provide a mechanism by which binding of EMPs to disrupted cells could inhibit proper barrier resealing. Support: Parker B. Francis Fellowship (Bauer and Moore), AHA #0765413B (N. Bauer) and 0765421B (T. Moore).
NATURALLY‐OCCURRING MISSENSE MUTATIONS IN THE CANINE CFTR GENE
Authors: Spadafora Domenico , Hawkins Eleanor C , Ballard Stephen T
Published: June 2021

Naturally‐occurring, cystic fibrosis‐causing mutations in the CFTR have not been identified in any non‐human animal species. Since domestic dogs are known to develop medical conditions associated with atypical CF in humans, (e.g. idiopathic bronchiectasis, pancreatitis, and aplasia of the vas deferens), we hypothesized that some dogs also carry CFTR mutations. Temporal temperature‐gradient gel electrophoresis (TTGE) was used to screen dogs for CFTR mutations. Canine whole blood was obtained from veterinary clinics and diagnostic laboratories. We screened DNA from 290 dogs that fell into one of three categories: dogs diagnosed with pancreatitis, dogs diagnosed with bronchiectasis, and dogs admitted to clinics for any illness (at‐large dogs). Thus far, 24 dogs have been identified with one of four missense mutations: R812W, P1281T, R1456W and P1464H. P1281T and P1464H mutations occur in relatively unconserved residues. R1456W is analogous to the human R1453W mutation, which has about 20% of normal CFTR function and is associated with pancreatitis and panbronchiolitis. R812W is in a highly conserved region of the regulatory domain. Mutation frequencies in pancreatitis and bronchiectasis animals were found at about twice the frequency of at‐large dogs. We conclude that naturally occurring CFTR mutations are relatively common in domestic dogs and can be detected with TTGE.

Supported by NIH HL633002 and Cystic Fibrosis Foundation grant BALLAR07G0
Mechanotransduction and signaling in wounded airway epithelial cells
Authors: Waters Christopher M. , Desai Leena P. , White Steven R.
Published: June 2021

Epithelial repair following injury to the airways occurs upon a substrate undergoing cyclic mechanical distention. The purpose of this study was to examine how mechanical stretch alters signaling pathways involved in wound healing of airway epithelial cells. We used a scratch‐wound model of human bronchial epithelial cells (16HBE) subjected to cyclic stretch (20% strain, 30 cycles/min) using the Flexercell device. As demonstrated previously, stretch caused inhibition of wound closure over 9 hr. We also found decreased PI3‐kinase activity, decreased membrane‐associated Tiam‐1, and decreased Rac1 activity in stretched cells. Adenoviral expression of a constitutively active form of PI3‐kinase stimulated wound closure of cyclically stretched cells, but not to the level of unstretched cells. Since stretch also decreased focal adhesion kinase (FAK) phosphorylation, we co‐expressed FAK, constitutively active PI3‐kinase, and JIP3 in stretched cells, and restored wound closure to the level of unstretched cells. Mechanical stretch alters multiple pathways that are important in restoration of epithelial integrity.

Supported by NIH grants HL064981 (CMW) and HL080417 (SRW).
Angiotensin II type‐1 receptor antagonism attenuates the hypercholesterolemia‐induced vascular dysfunction in mouse aorta
Authors: Kurtel Hizir , Granger D. Neil
Published: June 2021

Objective‐ Hypercholesterolemia induces a prothrombogenic and proinflammatory state that can be effectively reduced by the angiotensin II type 1 (AT1‐R) receptor antagonist losartan. The objective of this study was to determine the role for the AT1‐R in mediating the impaired endothelium‐dependent vasodilation (EDV) normally observed in the aorta of hypercholesterolemic mice. Methods and Results‐Vascular function was assessed in aortic rings by monitoring endothelium‐independent contraction to phenylephrine, endothelium‐independent dilation to sodium nitroprusside, and endothelium‐dependent dilation to acetylcholine (ACh). C57B1/6 mice were placed on a normal (ND) or high‐cholesterol (HCD) diet for 2 weeks. Separate groups of mice were treated with the AT1‐R antagonist losartan (25 mg/kg per day) in drinking water for 7 days. Serum cholesterol concentration in mice placed on the HCD was significantly higher than the mice placed on ND. EDV but not endothelium‐independent vasodilatation or constriction was significantly attenuated in aortic rings from HCD mice compared to their normocholesterolemic counterparts. The impairment in EDV to ACh observed after HCD was significantly attenuated by administration of losartan. Conclusions‐These data indicate that AT1‐R engagement contributes to the impaired EDV elicited by hypercholoesterolemia in mouse aorta. (Supported by HL26441)
Regional Difference of Increased Stiffness and Extra Cellular Matrix in Aging Monkey Aorta
Authors: Qiu Hongyu , Depre Christophe , Resuello Ranillo R.G. , Natividad Filipinas F. , Peppas Athanasios , Vatner Stephen F. , Shen You‐Tang , Vatner Dorothy E.
Published: June 2021

Aortic aneurysm is a vascular disease found most commonly in the elderly population and more frequently in the abdominal aorta (AA) compared to thoracic aorta (TA). However, the physiological basis underlying such regional differences is still under debate. Accordingly, we compared the mechanical and structural differences between TA and AA in aging (20‐25 years old, n=5) male monkeys by both physiological and histological measurements. Thoracic and abdominal aortic diameters were recorded directly from implanted miniature ultrasound crystals. Aortic stiffness was calculated from the pulse aortic pressure and diameter as the elastic modulus (K). TA and AA tissue sections were stained with picric acid sirius red for collagen and Verhoeff Van Gieson for elastin. Stiffness was significantly (p<0.05) higher in AA (K=2596±258) compared to TA (K=1870±195). Collagen density was also higher (40±1%) in AA vs TA (23±1%, p<0.05), whereas elastin density was lower in AA compared to TA (11.2±0.8% vs 21.8±0.7%, p<0.05). Therefore, in a non‐human primate aging model, the abdominal aorta, as compared with the thoracic aorta, demonstrates marked increases in stiffness which can be attributed to a greater increase in collagen and decreased in elastin. These differences in aortic architecture with aging may predispose the abdominal aorta to aneurysm formation.
Voluntary aerobic exercise abolishes age‐associated arterial stiffening in mice: relation to collagen subtype expression in the medial and adventitial layers
Authors: Fleenor Bradley S , Lesniewski Lisa A , Durrant Jessica R , Connell Melanie L , Donato Anthony J , Seals Douglas R
Published: June 2021

Voluntary aerobic exercise reduces large elastic artery stiffness with aging in humans, but it is unknown if reductions in collagen subtypes in the medial and/or adventitial layers of the arterial wall could be involved. Young (Y, 7 mo) and old (O, 31 mo) adult B6D2F1 mice underwent cage‐control (C) or voluntary wheel running (VR) for 12 weeks with carotid artery in vitro stiffness measures (n=9‐10/group) and immunohistochemical assessment of collagen (n=4‐8/group). VR (OVR: 3.7 ± 1.1 AU) abolished the age‐associated increase in passive stiffness in C animals (YC: 5.3 ± 0.8 vs. OC: 8.2 ± 1.0, P < 0.05). Collagen type I and III densities were 40‐55% greater in adventitia of OC vs. YC (P < 0.05), whereas OVR mice had medial and adventitial levels < OC (P < 0.05) and < YC. Collagen type IV was 30‐70% greater in media and adventitia of OC vs. YC (P < 0.05), whereas OVR had medial and adventitial levels < OC (P < 0.05) and < YC. Relative stiffness was not different in YC vs. YVR, and collagen expression was similar or lower in YVR. Short‐term voluntary aerobic exercise started late in life abolished the increase in carotid artery stiffness observed with sedentary aging in mice. Our data are the first to suggest a role for adventitial collagen deposition in age‐associated arterial stiffening, and that regular exercise may reduce stiffness in old mice in part by reducing collagen I, III and IV in the medial and adventitial layers of the arterial wall.

NIH AG013038, AG006537, AG015897, AG029337, AG000279
Psychological stress and endothelial dysfunction : a link between depression and vascular disease?
Authors: d'Audiffret Alexandre C. , Jackson Barbara , Riggs Dale , Insigrini Elsa , James Milinda E. , Nurkiewicz Timothy R. , Frisbee Jefferson C.
Published: June 2021

Depression is increasingly recognized as a risk factor for peripheral vascular disease in patients with no history of vasculopathy; as such this study evaluated vascular function in an established mouse model of chronic depression developed for behavioral research. 20 male Balb/cj mice were exposed to 6 weeks of unpredictable chronic mild stress (UCMS) to induce depression. The UCMS protocol consisted of random exposure to stressors including damp sawdust, social stress, exposure to predator sound/smell, reversal of the day/night cycle, and cage tilt. Depressive‐like behavior was evaluated using coat status/grooming in response to sucrose spray. Isometric tension development was assessed in aortic rings in response to acetylcholine and phenylephrine. Aortic responses to phenylephrine did not differ between groups, although acetylcholine‐induced relaxation was decreased in rings from mice exposed to the UCMS protocol. Parallel experiments indicated that acetylcholine‐induced NO generation was blunted in arteries of stressed mice versus controls. However, acetylcholine‐induced release of hydrogen peroxide was increased in mice receiving UCMS versus controls. These results may suggest that depression‐like behavior in mice is associated with an impaired vascular endothelial function and altered dilator signaling. The functional implications for these responses require further investigation.