bluuebunny/crossref_metadata_2025 huggingface dataset:
ACE2 ACTIVATION PROMOTES ANTITHROMBOTIC ACTIVITY
Authors: Fraga‐Silva Rodrigo Araujo , Sorg Brian S. , Wankhede Mamta , deDeugd Casey , Ferreira Anderson J. , Jun Yoo , Baker Matt B. , Li Y. , Castellano Ronald K. , Katovich Michael J. , Raizada Mohan K.
Published: June 2021

Hyperactivity of the axis ACE/AngII/AT1R of the renin‐angiotensin system is associated with occurrence of acute thrombotic event. Recently a novel concept of a counterrugulatory axis, ACE2/Ang‐(1‐7)/Mas, has emerged. We hypothesized that ACE2 would be protective against thrombosis. Thrombus was induced in the vena cava of SHR and WKY rats by FeCl3 solution. ACE2 and ACE protein expression and activities in the thrombus were determined by Western blot and fluorogenic kinetic assays, respectively. Real time thrombus formation was visualized by intravital microscopy of the vessels of nude mice. Ferric chloride‐induced thrombus weight was 40% higher in the SHR compared to WKY rats. This was associated with a 20% decreased in ACE2 activity in the thrombus of the SHR. In contrast, ACE2 protein expression and ACE activity did not differ between the thrombus of WKY rats and SHR. Inhibition of ACE2 by DX600 increased the thrombus weight by 30%, preferentially in the SHR. Furthermore, treatment with XNT resulted in a 30% attenuation of thrombus formation in both the SHR and WKY. In addition, XNT treatment prolonged the time for complete vessel occlusion and reduced thrombus size when observed under real‐time intravital microscopy. Our data demonstrated that a decrease in ACE2 activity is associated with increased thrombus formation in the SHR. Furthermore, activation of ACE2 attenuates thrombus formation.
Tamoxifen stimulates ROS production in human platelets via activation of NADPH oxidase: relationship to tamoxifen‐induced thrombosis
Authors: Dobrydneva Yuliya , Shah Vidhi , Vishneski Susan
Published: June 2021

Tamoxifen is a widely prescribed drug for the treatment and prevention of breast cancer; however, thrombosis is a well‐known adverse effect of tamoxifen. Previously we established that tamoxifen promotes calcium influx into platelets, a critical step in platelet activation. In this study we sought to determine the mechanism of tamoxifen‐induced platelet activation. Experiments were conducted with freshly isolated human platelets from healthy volunteers. Tamoxifen increases binding of Annexin V and PAC‐1 antibody, surface markers of platelet activation. Tamoxifen promotes production of superoxide, as measured by lucigenin chemiluminescence; NADPH oxidase is likely the source of superoxide. Tempol and Tempo (ROS scavengers), apocynin and apocynin dimer (NADPH oxidase inhibitors) and Ly294002 and wortmannin, (inhibitors of PI3 kinase, upstream effector of NADPH oxidase) block tamoxifen effect. Raloxifene, a tamoxifen analog, does not cause platelet activation or superoxide production. This could explain lower incidence of thrombotic events in patients taking raloxifene compared to tamoxifen. Funded by Commonwealth Health Research Board, Cancer Research and Prevention Foundation, and American Heart Association.
Augmented angiotensin II‐induced arteriolar constrictions in mice with type 2 diabetes mellitus ‐ role for cyclooxygenase‐2
Authors: Bagi Zsolt , Koller Akos , Kaley Gabor
Published: June 2021

Inhibitors of the renin angiotensin system have beneficial vascular effects in diabetes mellitus. Thus we hypothesized that angiotensin II (Ang II) elicits augmented arteriolar constriction in mice with type 2 diabetes (db/db mice) is due to factor(s) produced by the increased activity and expression of cyclooxygenase‐2 (COX‐2). We found that isolated, pressurized gracilis arterioles of db/db mice exhibited enhanced constrictions to Ang II (10‐11‐10‐8 M) compared to controls (db/db max: 82±4%, control max: 59±2%, P<0.05). Constrictions to norepinephrine (NE) were similar in the two groups (db/db max: 68±4%, control max: 72±2 %). Inhibition of nitric oxide synthesis with L‐NAME did not affect Ang II‐ and NE‐induced constrictions in arterioles of control and db/db mice. In arterioles of db/db mice the enhanced constrictions to Ang II were reduced by the selective COX‐2 inhibitor, NS‐398 (max: 62±3%) or by the EP1 receptor antagonist, AH‐6809 (max: 57±4%) to the levels observed in control mice. Also, in gracilis muscle arterioles of db/db mice an increased COX‐2 protein expression was detected by immunohistochemistry. Thus, we propose that in type 2 diabetes there is an increased activity and expression of COX‐2, a mechanism by which Ang II stimulates the production of PGE2, which via activating EP1 receptors augments constrictions of arterioles to Ang II. (AHA 0855910D, 0735540T and NIH PO1‐HL‐43023)
Development of a predictive model for negative microvascular outcomes in the metabolic syndrome
Authors: Frisbee Jefferson C. , Goodwill Adam G. , James Milinda E. , Brock Robert W. , Hollander John M. , Frisbee Stephanie J.
Published: June 2021

Evolution of the metabolic syndrome is a multi‐factorial process with a constellation of pathologies developing over time. Consequently, poor vascular outcomes in the metabolic syndrome are also temporally distributed, and an integrated understanding of these outcomes requires information regarding their sequence of development. To address this, we examined development of the metabolic syndrome, systemic disease biomarkers, and negative vascular structural/functional outcomes in obese Zucker rats at 6‐7, 9‐10, 12‐13, 15‐16 and 19‐20 weeks. For these initial procedures and model development, no interventional strategy against either the metabolic syndrome or the poor outcomes was employed. Prior to development of vascular dysfunction, endothelial arachidonic acid metabolism was altered toward TxA2 production; predicted by insulin resistance. This outcome was followed by an increase in markers of systemic inflammation, resulting in systemic oxidant stress and reduced vascular NO bioavailability. Immunohistochemistry suggested an increased venular adhesion marker expression (ICAM‐1, VCAM‐1) associated with the loss of NO bioavailability and this contributed significantly to a reduction in microvessel density. Taken together, these data begin to provide a temporally relevant, predictive model for poor microvascular outcomes in the metabolic syndrome. (NIH R01 DK64668, AHA EIA 0740129N)
Hyperglycemia‐induced mitochondrial dysfunction and oxidant generation in mouse renal microvascular endothelial cells is reversed by C‐peptide
Authors: Vejandla Himani L. , Frisbee Jefferson C. , Dabkowski Erinne R. , Hollander John M. , Kothur Anand , Brock Robert W.
Published: June 2021

Endothelial cells are prime targets for damage during diabetes because the glucose transport rate of these cells does not decline quickly enough, leading to intracellular hyperglycemia. Eventually this increases the voltage gradient across the mitochondrial membrane, reaches a threshold and generates oxidants. The goal of this study was to determine the impact of C‐peptide on mitochondrial membrane potential (Δψm), respiratory function and oxidant production in renal microvascular endothelial cells (RMECs) exposed to high glucose. To accomplish this, RMECs were exposed to high (25 mM) or low glucose (5 mM with raffinose) in the absence or presence of C‐peptide (6.6 nM) under non‐premissive conditions. High glucose exposure resulted in significant mitochondrial hyperpolarization with significant oxidant generation. Concomitant treatment with C‐peptide restored Δψm with no apparent oxidant generation (p<0.002). Moreover, high glucose cells displayed significantly lower state 3 respiration rates (p< 0.05) compared to those in the low glucose condition. C‐peptide treatment restored state 3 rates to normal (p< 0.05). Respiratory control ratios were significantly higher in the high glucose cells treated with C‐peptide than those in high glucose alone (p<0.001). Together, these data suggest that C‐peptide protects against endothelial mitochondrial dysfunction during hyperglycemia. NIH RO1 DK067582
Acute vascular changes in renal arterial anatomy after cryosurgery
Authors: Spaan Jos , Lagerverld Brunolf , van Horssen Pepijn , van den Wijngaard Jeroen , de la Rosette Jean , Wijkstra Hessel
Published: June 2021

Introduction

Cryosurgery is gaining importance as an optional curative or palliative treatment technique for urological cancers. The aim is to indicate which part of the microcirculation is acutely destroyed by this technique.

Methods

In 6 anesthetized pigs kidney cryoablation was applied for 20 minutes using a triple needle probe configuration (Cryoneedle TM). The kidneys where allowed to thaw in situ and then excised., its main artery was flushed with saline and filled with a fluorescence dyed elastomere. The kidney was, frozen and processed in an imaging cryomicrotome. After each slice the fluorescence in the cutting plane was measured applying a 2000 by 2000 pixel digital camera. 3 D image processing revealed information about arterial structure down to 20 μm vessels. As a statistical measure the summed length, Ld of all segments having a diameter, d, were summed for regions of interest, ROI.

Results

The ablated zones were clearly noticeable in the white light and 3‐D fluorescent images. The histograms of Ld versus d as well as dPeak (d where Ld is maximal) where independent of the selection of non‐ablated ROI. For ablated areas dPeak shifted to larger diameters by 70 microns and Ld at dPeak without ablation reduced by a factor of 10.

Conclusion

After cryoablation, acute injury of the renal arterial vessels induced destruction of the vessels smaller than 200 μm leaving the structure of larger vessels intact.
A vasoprotective role for free‐radical scavenger therapies in chronically hyperglycemic mice
Authors: Savery Michele D , Jiang John X , Damiano Edward R
Published: June 2021

The presence of a hydrodynamically relevant endothelial‐cell surface glycocalyx, ~ 0.5 µm thick, is well established under normal physiological conditions. This layer is partially degraded by light‐dye treatment and completely degraded following hyaluronidase treatment. Many complications of diabetes are derived from vascular disease, and the glycocalyx is situated as the first line of defense against vascular damage. Therefore, we seek to establish the effects of hyperglycemia on the glycocalyx and the effectiveness of various therapies. We analyzed data obtained in venules of chronically hyperglycemic NOD mice receiving subsistence levels of insulin therapy, half of which were also treated with synthetic catalytic free radical scavengers (SOD). In mice receiving insulin only, we observed that the glycocalyx thickness is degraded to ~ 0.21 µm (n = 10), compared with ~ 0.48 µm (n = 13) in healthy wild type and NOD mice. This degradation is significant (p < 0.05) and is comparable to that seen after free‐radical damage. In mice also treated with SOD, the glycocalyx thickness was ~ 0.39 µm (n = 10); this is not significantly different from the healthy control group. Furthermore, we seek to link glycocalyx degradation to vascular pathology. Preliminary data suggest that chronic enzymatic degradation results in pathological levels of microalbuminuria, an early indicator of kidney dysfunction due to vascular damage.
Ribonucleotide reductase in an anoxia tolerant vertebrate, the crucian carp
Authors: Sandvik Guro Katrine , Nilsson Goran Erik
Published: June 2021

Can enzymes that are oxygen‐dependent in all known eukaryotes, function in the anoxic crucian carp (Carassius carassius)? The crucian carp can survive for several months in anoxia, partly because of its extraordinary ability to convert lactate to ethanol, which allows it to avoid lactate accumulation. Previous studies have shown that crucian carp cells can undergo cell division in anoxia. However, DNA replication should not be possible in anoxia, because in all eukaryote organisms examined ribonucleotide reductase (RNR), which produces the building material for DNA, is dependent on oxygen to form a catalytically important radical. Therefore, the RNR in crucian carp must be exceptional in some way, possibly by forming an unusually stable radical, or by having an exceptionally high oxygen affinity. We have cloned and sequenced several RNR variants from crucian carp. Interestingly, quantitative real‐time PCR measurements in normoxic and anoxic crucian carp revealed that the expression of the small RNR subunit (R2), which is the subunit activated by oxygen, was maintained in anoxia. RNR proteins were in‐vitro expressed in E. coli for structural and enzymatic studies. Amino acid analysis and EPR spectroscopy suggest that the variants are structurally very similar to mammalian RNR, but ongoing studies will show if their oxygen affinity are different. Supported by the Research Council of Norway.
Non‐uniform inter‐ and intra‐fiber strain observed in MRI studies of contracting medial gastrocnemius
Authors: Sinha Shantanu , Shin David , Hodgson John , Chi Sheng‐Wei , Chen Jiun‐Shyan , Edgerton Reginald V
Published: June 2021

Finite element models of contracting muscle suggest non‐uniform strain along individual muscle fibers which is dependent upon the location of the fibers within a muscle. We have investigated the distribution of strain along muscle fibers in different regions of the medial gastrocnemius muscle using phase contrast magnetic resonance imaging. The greatest changes in fiber strain occurred in the proximal region of the superficial aponeurosis and the distal region of the deep aponeurosis. These regions are theoretically the regions of highest stress concentration as the muscle narrows near its ends. The lowest strains occurred at the opposite ends of the same fibers. Fibers in the mid‐region of the muscle exhibited intermediate strains with a more even distribution of strain along the fibers. The ratio of aponeurosis displacement to fiber length change (gear ratio) was generally greater than unity, indicating that the aponeurosis moved more than the muscle fibers shortened. There was a significant dependence on the proximo‐distal position (p<0.001) with a progressive increase in the ratio moving proximally (p<0.05). There was no significant effect of location on overall fiber length change. Differing regional changes in the separation of aponeurosis and/or to regional differences in pennation angles may be responsible for changes in gear ratio. Funded by NIAMS Grant RO1 AR‐53343.
Impaired renal blood flow autoregulation and renal damage in a mouse model of reduced βENaC
Authors: Grifoni Samira de Campos , Chiposi Rumbidzayi , Drummond Heather
Published: June 2021

Previous studies from our laboratory indicate β Epithelial Na+ Channel (βENaC) mediates myogenic constriction in small renal arteries. Although studies suggest myogenic constriction contributes to renal blood flow (RBF) autoregulation (AR) and protects the kidney from injury, the importance of myogenic constriction in AR and renal injury has not been tested. Thus, the aim of this study was to test the hypothesis that suppression of βENaC impairs RBF‐AR and leads to renal damage. We evaluated steady state changes in RBF in response to step increases in renal perfusion pressure (RPP) in anesthetized mice with reduced levels of βENaC (m/m) and littermate controls (+/+ and +/‐). The autoregulatory index (AI) was calculated as: AI=[(RBF2‐RBF1)/RBF1]/[(RPP2‐RPP1)/RPP1]. AR to step changes in RPP was impaired in m/m compared with control mice, as indicated by higher AI (m/m=1.8±0.2, n=4 vs. control=0.2±0.07, n=3). 24‐hour urine albumin excretion and macrophage infiltration, markers of renal injury, were assessed. Urinary albumin excretion was higher in m/m (12.8 [0]± 1.4 μg/day, n=13) than in +/+ mice (9.3 ± 1.2, n=9, p=0.04). Furthermore, macrophage infiltration was higher in the renal cortex of m/m vs. +/+ mice (33 ± 1.4 cells, n=3; 9.3 ± 1.1, n=3, respectively). These data suggest that reduction in βENaC expression impairs RBF‐AR and may predispose the kidney to hypertension‐related injury. Supported by: NIH‐NHLBI, AHA
Aldosterone induces sympatho‐excitation via brain mitogen‐activated protein kinase signaling pathways in rat
Authors: Zhang Zhihua , Yu Yang , Wei Shunguang , Felder Robert B.
Published: June 2021

Recent evidence shows that brain mitogen‐activated protein kinase (MAPK) signaling plays a critical role in mediating renin‐angiotensin system influences on sympathetic drive. We tested the contribution of brain MAPK to aldosterone‐induced sympatho‐excitatory responses. In anesthetized rats, we recorded renal sympathetic nerve activity (RSNA), mean blood pressure (MBP) and heart rate (HR) at baseline and for 4 hours during intravenous (IV) infusion of aldosterone, with or without concomitant intracerebroventricular (ICV) infusion of the mineralocorticoid (MR) antagonist RU28318 or selective MAPK inhibitors. Hypothalamic tissues were then collected for molecular analysis. Aldosterone infusion increased (p<0.05) RSNA, HR and MBP, as well as the hypothalamic expression of phosphorylated (relative to total) p44/42 and p38 MAPK. Concomitant ICV infusion of RU28318 blocked phosphorylation of p44/42 MAPK, blunted phosphorylation of p38 MAPK, and prevented the increases in RSNA, HR and MBP. The p44/42 MAPK inhibitor PD98059 reduced the aldosterone‐induced RSNA, HR and MBP responses, and the p38 MAPK inhibitor SB203580 reduced the aldosterone‐induced HR and MBP responses. These data suggest that aldosterone, originating in the periphery and acting on brain MR, activates brain MAPK pathways to produce sympatho‐excitatory responses. Supported by a VA Merit Review Award.
Endothelins‐noradrenergic transmission interaction in the olfactory bulb of Doca‐salt rats
Authors: Abramoff Tamara , Nabhen Sabrina , Hope Sandra , Guil Maria , Bianciotti Liliana , Vatta Marcelo
Published: June 2021

We have previously reported that endothelins (ETs) modulate noradrenergic transmission in the olfactory bulb (OB) of normotensive rats. As it has been suggested that ETs are involved in the development and/or maintenance of hypertension we sought to establish the effect of exogenous ETs on tyrosine hydroxilase (TH) activity and expression in the left and right OB of DOCA‐salt rats. Results were expressed as percentage of control group, and statistical analysis was performed by two‐way ANOVA and SNK. p values of 0.05 or less were considered significant. Results showed that in the right OB of DOCA‐salt rats TH activity and expression were increased by 40% and 145%, respectively (p<0,001, n=8). In the left OB TH expression was unaltered but its activity was increased by 25% (p<0,001, n=8). When right and left OBs of DOCA‐salt rats were incubated with 10nM ET‐1 or ET‐3, TH activity was reduced by 31% in the right OB (p<0,001, n=8) but not in left OB. These findings show that TH activity and expression were different in DOCA‐salt rats as compared with control rats. Furthermore, ETs exerted distinct effects on TH in the right and left OB, supporting an asymmetric functionality as reported in other brain areas, but the significance of this finding is presently unknown. This study suggests that ETs may be involved in the development and/or maintenance of hypertension in this animal model. (UBACyT B079, CONICET‐PIP 5929, ANPCyT PICT‐38333)
The effects of post stroke Captopril versus Losartan treatment on cerebral blood flow autoregulation after hemorrhagic stroke
Authors: Smeda John Stanislaw , Daneshtalab Noriko
Published: June 2021

Kyoto Wistar stroke prone hypertensive rats (SHRsp) lose the ability to regulate constant cerebral blood flow (CBF) in response to varying mean arterial pressures (MAP) after hemorrhagic stroke (HS). We tested the ability of Captopril (CAP) and Losartan (LOS) treatment to restore CBF autoregulation after stroke. CAP (50mg/kg/day) or LOS (35 mg/kg/day) were administered orally at the first signs of stroke. Laser Doppler techniques measured alterations in CBF in response to varying MAP (90 to 300 mmHg). Both post stroke treatments expanded the lifespan of SHRsp from 14 to > 60 days without greatly altering blood pressure. Two weeks before HS, SHRsp regulated near constant CBF up to an MAP of 200 mmHg. CBF regulation was lost at the first symptoms of stroke. CBF (relative to maximal flow) increased linearly with MAP and was 2 fold higher than pre‐stroke levels. Post stroke CAP and LOS treatment restored CBF regulation to pre‐stroke levels within 12 days. However, CAP treated SHRsp lost the ability to autoregulate CBF after 32 days of treatment whereas LOS treated rats retained this function to 60 days. It is possible that the ability of angiotensin II to stimulate AT‐2 receptors under conditions of AT‐1 receptor blockade by LOS may be of advantage in producing a more permanent restoration of cerebrovascular function after HS.
Sickle Cell transgenic mice demonstrate a pro‐thrombogenic phenotype
Authors: Gavins Felicity n.e. , Russell Janice , Granger D Neil
Published: June 2021

In this study we investigated the mechanisms involved in thrombus formation in sickle cell disease (SCD) because SCD patients are more susceptible to thrombotic events, with unknown reason.

Thrombosis was induced using the light/dye method in cerebral venules and arterioles of wild‐type (WT:C57Bl/6), sickle cell transgenic (SCD; βs) and WT/βs chimeric (positive control) mice (1). Flow cessation time was recorded.

Thrombus formation was reduced by 50% in SCD mice vs. WT in venules and arterioles, but not observed in chimeric mice.

The clinical implication of anti‐coagulant therapy was investigated, using Hirudin and anti‐thrombin III (AT3) inhibitor. Hirudin increased thrombus formation in SCD mice, but only in WT venules, suggesting a possible heightened sensitivity in SCD microvasculature. The AT3 inhibitor increased flow cessation in all vessels of WT and SCD mice, implicating the importance of all coagulation pathways.

This study further supports the growing evidence for a pro‐inflammatory and pro‐thrombogenic microcirculation in SCD mice, in particular the notion that SCD promotes thrombus formation. Understanding the susceptibility and pathways involved in thrombogenic situations that occur in SCD, it may lead to the identification of potential therapeutic targets in this painful and life‐threatening disease.

Supported by the BBSRC

1. Wood, K. et al. AJPHCP. 2004.
Preventing Increased Blood Pressure Improves Severity of Stroke, Vascular Remodeling, and Myogenic Tone in the Obese Zucker Rat
Authors: Osmond Jessica M. , Mintz James D. , Stepp David W.
Published: June 2021

Obesity is a risk factor for stroke, but the underlying mechanisms by which obesity increases stroke risk are unknown. Hypertension is a major risk factor for stroke. We have previously reported that obese Zucker rats (OZR) have a worse stroke outcome and display evidence of remodeling and increased myogenic tone of the middle cerebral artery (MCA) in parallel with hypertension compared to lean controls (LZR). This study tested the hypothesis that preventing the increase in blood pressure in OZR would improve remodeling, tone, and the outcome of stroke. Blood pressure was measured by telemetery in LZR, OZR, and OZR+hydrochlorthiazide (HCT, 2 mg/kg/day) from the age of 6 to 16 weeks. Vessel structure and function were assessed in isolated, pressurized MCAs. Cerebral schemia was induced for 60 minutes using an intralumenal suture technique, followed by 24 hours of reperfusion. HCT treatment effectively prevented the increase in blood pressure in OZR. Additionally, HCT prevented the changes in MCA structure and tone observed in untreated OZR. Importantly, infarct size was normalized in OZR+HCT following ischemia/reperfusion. These results indicate that increased pressure is essential for driving the changes in infarct size and MCA structure and function in OZR. These observations suggest that increased blood pressure evident in obese individuals is a necessary major determinant for stroke risk in this population.
Enhanced Carbachol‐Mediated Relaxation in Aorta of Mice Lacking the Potassium Channel TWIK‐2
Authors: Lloyd Eric Edward , Namiranian Khodadad , Marrelli Sean P , Bryan Robert M
Published: June 2021

TWIK‐2, a member of the Two‐Pore Domain K channel family, is highly expressed in the vascular system, however the function is unknown. We hypothesized that TWIK‐2 is involved with regulating vascular tone in arteries and tested this hypothesis on animals lacking TWIK‐2 (KO). Body composition analyses and blood pressures were similar in the KO and wild type (WT) mice. Isolated arteries were evaluated by wire myography. The tension in response to 60 mM KCl was enhanced in the TWIK‐2 KO. However, constrictor responses to phenylephrine and U46619, a thromboxane A2 mimetic, were similar. Relaxation to carbachol, an acetylcholine analog, was measured after precontraction with phenylephrine (0.5 μM). Arteries from KO animals showed that dose dependent relaxations to carbachol were enhanced by 37% (n=5, p<0.01). The NO‐synthase inhibitor, L‐NAME (100 μM), abolished the relaxations to carbachol in both strains. In summary, our data indicates that TWIK‐2 does have a role in regulating arterial tone. Additional studies are ongoing to determine the specific mechanism of TWIK‐2 in regulating vascular tone.

This work was supported by research grants R01 NS 46666 and NS 038660.
Inhibition of integrin aVB3 prevents uPA–mediated impairment of cerebrovasodilation after cerebral hypoxia/ischemia
Authors: Armstead William M , Kiessling J Willis , Cines Douglas B , Higazi Abd Al‐Roof
Published: June 2021

Cerebral hypoxia/ischemia (H/I) impairs cerebrovasodilation in response to hypercapnia and hypotension in the newborn pig, exogenous uPA potentiates this effect, while blockade of endogenous uPA‐mediated vasoactivity prevents it completely. This study investigated the role of integrin αVβ3, in uPA‐mediated impairment of cerebrovasodilation after H/I in piglets equipped with a closed cranial window. Pial artery dilation induced by hypercapnia (pCO2 75 mm Hg) and hypotension (mean arterial blood pressure decreased by 45%) was blunted after H/I, reversed to vasconstriction in piglets treated with uPA (10−7 M), a concentration observed in CSF after H/I, but reverted to dilation no different than pre‐insult in piglets administered an anti αVβ3 antibody (10 ng/ml) in addition to uPA (26 ± 1, 9 ± 1, ‐10 ± 3, and 22 ± 3 % for hypercapnia before H/I, after H/I, after H/I with uPA, and after H/I with combined uPA and anti‐αVβ3 antibody). Responses to isoproterenol were unchanged after H/I and combined uPA and anti αVβ3 antibody. Similar results were obtained for combined administration of uPA with the αVβ3 antagonist cRGDfV and RGDS, but not for the inactive analogue, RGDES. These data show that activation of the integrin αVβ3 contributes to uPA‐ mediated impairment of pial artery dilation after H/I. These data suggest that inhibition of uPA and integrin signaling may preserve cerebrohemodynamic control after H/I.
Correlation between acute stroke and microalbuminuria. Potential role of underlying systemic microvascular endothelial disease
Authors: Toth Peter , Koller Akos , Marko Lajos , Halmai Richard , Bosnyak Edit , Szapary Laszlo , Komoly Samuel , Nagy Judit , Wittmann Istvan
Published: June 2021

We hypothesized that ischemic stroke is a manifestation of a systemic microvascular endothelial disease, which is thought to be characterized by microalbuminuria (albumin/creatinine: male patients: 2.5‐25; female patients: 3.5‐35 mg/mmol). Thus in patients with acute ischemic stroke (n=100) HPLC was used to measure urinary albumin/creatinine and glomerular filtration rate was estimated (eGFR) by the MDRD formula to characterize kidney function. To assess the severity of stroke the Scandinavian Stroke Scale (SSS) and the National Institute of Health Stroke Scale (NIHS) were used. We have found that 54.1% of stroke patients with normal kidney function (eGFR above 90 ml/min/1,73m2) had microalbuminuria. In addition, 69% of stroke patients with microalbuminuria had decreased kidney function, as well (eGFR below 90 ml/min/1,73m2). The severity of microalbuminuria correlated significantly (p<0,01) with the severity of stroke (SSS: NM: r = ‐0,38; HPLC: r = ‐0,35; and NIHS: NM: r = 0,24; HPLC: r = 0,24). We suggest that both ischemic stroke and microalbuminuria are likely a consequence of a systemic vascular endothelial disease and thus it seems to be important to assess microalbuminuria and kidney function in all stroke patients.

Grants: Hungarian Sci. Res. Funds/OTKA T48376, K71591, F68638; Health Sci. Council/ETT 364/2006, Hungarian Neuroimaging Foundation, Am. Heart Assoc. Founders Aff. 0855910D, USA
Real‐time imaging of Hsp27‐cfp redistribution in response to oxidative stress
Authors: Martin Jody Lee , Carbajal Luis
Published: June 2021

The small heat shock protein, hsp27, has been suggested to regulate actin cytoskeleton structure and modulate the interaction of actin and myosin, given its biochemical properties and high expression levels in smooth muscle cells (SMCs) under stress. This may contribute to the mechanical plasticity of SMCs. It is proposed that hsp27's putative interaction with the actin cytoskeleton and its subcellular localization is regulated by its phosphorylation via the p38 MAP kinase pathway (MAPK). This pathway is activated by mechanical and oxidative stress, like hydrogen peroxide, and can be specifically inhibited with SB203580 (SB). We have established a real‐time oxidative stress smooth muscle cell imaging protocol utilizing time lapse microscopy to examine cellular redistribution of hsp27‐cfp in response to 200μM hydrogen peroxide +/‐ SB. Immunoanalysis of endogenous and ectopically expressed hsp27 demonstrates its phosphorylation and p38 activation. Interestingly, Hsp27‐cfp translocated to the perinuclear region with oxidative stress only with SB treatment. These results illustrate the dynamic and complex nature of hsp27 in the oxidative stress response of vascular smooth muscle cells.
The effects of exercise on caspase independent endonucleaseG and apoptosis inducing factor with regards to age‐related apoptosis
Authors: Courtney Sean Michael , Kim Jong‐Hee , Macias Brandon R , Canon Claire N , Lawler John M
Published: June 2021

Exercise may reduce oxidative stress, mitochondrial dysfunction, and mitochondrial caspase‐dependent apoptosis with age. However, it is unclear whether the caspase‐independent apoptosis via EndonucleaseG (EndoG) and Apoptosis Inducing Factor (AIF) is affected by exercise in aging skeletal muscle. We hypothesize that exercise will attenuate apoptosis brought on by the translocation of EndoG and AIF from the mitochondria to the nucleosome in aging white gastrocnemius. Twenty‐four Fischer Brown Norway rats were randomly assigned to four groups, young sedentary, old sedentary, young exercisers and old exercisers. In the soluble fraction there was an 86.4 % increase in the old sedentary group in EndoG, but there was no change in the young groups. EndoG protein level in the nucleosome fraction of young exercisers decreased 49 % and old sedentary increased by 86.5% when compared to young sedentary controls. The data indicated that exercise was protective against apoptosis by decreasing the translocation of EndoG to the nucleosome in aged skeletal muscle.
NFATc3 is required for chronic hypoxia‐induced pulmonary hypertension: role of BKα and β subunits
Authors: Bierer Ryann , de Frutos Sergio , Nitta Carlos H , Resta Thomas C , Friedman Jessica , Gonzalez Bosc Laura
Published: June 2021

Chronic hypoxia (CH)‐induced pulmonary arterial remodeling and right ventricular (RV) hypertrophy require NFATc3 activation. Hence, we hypothesized that NFATc3 is also required for CH‐induced pulmonary hypertension. Mean RV pressure (MRVP mmHg) was measured in anesthetized wild type (WT) mice treated with cyclosporine A (CsA, 25 mg/Kg/day, NFAT inhibitor) or vehicle (V)‐containing food, and in NFATc3 heterozygote (HET) and knockout (KO) mice exposed to normoxia (N) or CH (380 mmHg; 21 days). CsA significantly attenuated CH‐induced increases in MRVP.

CH increased MRVP in WT and HET but not in KO mice (NWT: 11.8±2.3; NHET: 10.4±1.8; NKO: 15.5±3.6; CHWT: 20.0±4.4*; CHHET: 16.1±4.7*; CHKO: 17.8±5.9; n=5‐10; *p<0.05 vs. N).

BK channels regulate membrane potential and contractility, and NFATc3 regulates its expression in smooth muscle. A reduction in BKα and/or ? expression in pulmonary arteries via NFATc3 could underlie the increased pressure. There was no change in BK ? expression. KO mice showed less BKα mRNA than WT in N. As expected, CH induced a decrease in BKα that was not different from CHKO. Consistently, NFAT activation induced an increase in BKα promoter activity in human PASM cells that was prevented by CsA. This response was lost in cells exposed to 2% O2 for 24 h but basal activity was reduced compared to N. These data demonstrate that NFATc3 is required for CH‐induced pulmonary hypertension and basal BKα mRNA expression. In addition, a reduction in BKα mRNA may be involved in the development of hypertension independently of NFATc3. 1R01HL088151‐01A1/NIH.
The magnitude of ventilatory long term facilitation and the hypoxic ventilatory response is affected by time of day
Authors: Carney L M , Anthouard M N , Lee D S , Badr M S , Mateika J H
Published: June 2021

Purpose

To determine if the magnitude of ventilatory long term facilitation (vLTF) and the hypoxic ventilatory response (ΔVE/ΔSaO2 ‐ HVR) following exposure to intermittent hypoxia (IH) is affected by time of day.

Methods

7 male subjects with obstructive sleep apnea were exposed to IH daily for 11 days. The protocol included 12 4‐minute episodes of hypoxia (PETO2 ‐ 50 mmHg, PETCO2 ‐ 3 mmHg above baseline) followed by a 30 minute end‐recovery period. On days 1 and 11 IH was applied in the evening (PM), and in the morning (AM) on days 2‐10. Before and after daily exposure to IH the HVR was measured.

Results

On Day 1 & 11 (PM) and Day 2 & 10 (AM), VE and the HVR were significantly greater after IH compared to baseline, consistent with our other findings (data not shown ‐ see abstracts from Gerst et al. and Lee et al.). However, vLTF was less (p = 0.004) and the HVR was greater (p = 0.01) during the AM compared to the PM studies before (Days 1 & 2) and after (Days 10 vs. 11) 11 days of IH. Additionally, the vLTF and HVR responses after IH were greater on Days 10 & 11 compared to Days 1 & 2 (vLTF ‐ p = 0.01; HVR ‐ p = 0.05).

Conclusions

The magnitude of vLTF is less and the HVR is greater after AM compared to PM exposure to IH. This relationship persists following chronic IH.

Funding

NIH (HL‐085537)
Immunohistochemical identification of K+ channel subunits in neurons from chemosensitive regions of rat brainstem
Authors: Putnam Robert W. , Martinez Catalina , Nichols Nicole L.
Published: June 2021

CO2‐sensitive neurons control breathing and are found in various regions of the brainstem. Neurons within these areas respond to CO2 with different percentages and different magnitudes of changes in firing rate responses to CO2. We propose that neuronal responses to CO2 are different because of differences in the ion channels expressed on neurons in these regions. The current study localized pH‐sensitive K+ channels in two different areas of the brainstem, the solitary tract (SC) (nucleus tractus solitarius, NTS, and dorsal motor nucleus, DMN) and locus coeruleus (LC). Brainstem slices (300µm) were fixed and thin sectioned (20‐40µm). Nissl staining was used to stain all cells and glial fibrillary acidic protein (GFAP)stained astrocytes. Neurons were identified as Nissl positive/GFAP negative. Sections were stained with antibodies for pH‐sensitive subunits of voltage‐sensitive (Kv1.4) and inwardly‐rectifying (Kir2.3 and Kir4.1) K+ channels. We found that Kv1.4 was prevalent on SC neurons (60‐90%), which stained less for Kir2.3 and 4.1 (30‐40%). In contrast, LC neurons stained similarly (50‐60%) for Kv1.4, Kir2.3 and Kir4.1. These data are consistent with an A current (inhibitable by 4 aminopyridine) playing a major role in the CO2 response of SC neurons but both an A current and Kir channels playing a role in the CO2 response of LC neurons.

Supported by NIH R01 HL56683 (RWP) and T35 HL007805 (CM).
The time domains of the ventilatory responses of bowfin (Amia calva) to prolonged hypoxia
Authors: Ciuhandu Cosima , Chan Alison , Milsom William K
Published: June 2021

The acute responses of fish to hypoxia are well established, however the time course for the hypoxic ventilatory response in chronic hypoxia is not well understood. In the present study we assessed the ventilatory and cardiovascular responses of the facultative air breather, Amia calva, to severe hypoxia for 3 days. During prolonged exposure to hypoxia, there was an initial increase in gill breathing frequency, amplitude and total gill ventilation over the first three hours of hypoxic exposure, but this was not sustained. After the initial acute response, there was a decrease in total ventilation that lasted until about 24 hours of hypoxic exposure, when a secondary increase in ventilation started taking place. There was also an immediate bradycardia that was not sustained. These changes were accompanied by changes in the size, and density but not distribution of putative gill neuroepithelial cells (homologous to carotid body glomus cells) after 3 days of hypoxic exposure indicating the importance of these cells in the hypoxic ventilatory response. The time domains seen in gill breathing in Amia are similar to those reported in mammals however the time course of these responses is slower with a ventilatory "roll off" developing in hours not minutes. (funded by NSERC of Canada)
Hypoxic ventilatory depression is attenuated by blockade of K(ATP) channels and adenosine receptors in the isolated bullfrog brainstem
Authors: Hedrick Michael S. , Chen Anna K.
Published: June 2021

This study examined the role of K(ATP) channels and adenosine receptors in regulating the hypoxic ventilatory response in isolated brainstems of post‐metamorphic bullfrogs (Lithobates catesbeiana). We used an in vitro brainstem preparation from post‐metamorphic frogs (N=12; T‐K stages >25) that was superfused with oxygenated artificial CSF (20‐22 ºC) and generated spontaneous respiratory motor output measured by suction electrodes attached to cranial nerves (CN) V, X and XII. Control brainstems superfused with hypoxic artificial cerebrospinal fluid (aCSF) bubbled with 98% N2/2% CO2 produced a ventilatory depression marked by significant reductions in lung burst frequency within 15‐30 min that were sustained for 3 h of hypoxia. Reoxygenation with 98% O2/2% CO2 returned lung burst frequency to control levels. Hypoxic brainstems superfused with aCSF containing the K(ATP) channel blocker, 2,3‐butanedione monoxime (BDM;500 μM) and the adenosine receptor antagonist, theophylline (100 μM), did not exhibit a hypoxic ventilatory depression until 90 min after the onset of hypoxia, but returned to control levels upon reoxygenation. These data suggest that the hypoxic ventilatory depression observed in the isolated bullfrog brainstem is mediated in part by K(ATP) channels and/or adenosine receptors. Supported by NIH SO6 GM48135.
eNOS induced angiogenesis is blocked by the tyrosine kinase inhibitor Vatalanib (PTK787) in a normoperfused rodent model
Authors: Stone Oliver Andrew , Harper Steven J , Bates David O
Published: June 2021

Therapeutic angiogenesis has been largely unsuccessful to date. We have shown recently that a combination of growth factor overexpression (Ad.VEGF and Ad.Ang‐1) and physical forces (Ad.eNOS or Prazosin) results in a mature neovasculature (Benest et al 2008). To further investigate the mechanisms of eNOS induced angiogenesis, eNOS was overexpressed in the adult rat mesentery and intravital and confocal imaging used to examine vessel morphology following treatment with PTK787. PTK787 significantly inhibited the eNOS induced increase in functional vessel area (32±4 vs 917±80 vs 159±45%, P<0.001) and vessel density (321±18 vs 549±45 vs 336±25mm−2, P<0.01). In contrast, eNOS in the presence of PTK787, stimulated an increase in conduit vessel density (16‐35μm:63±8 vs 153±31 vs 150 ±16mm−2, P<0.05) and a reduction in exchange vessel density (<16μm:234±17 vs 186±25 vs 397±67 mm−2, P<0.001), resulting in a skewed vessel distribution. Most strikingly, eNOS + PTK787 resulted in the recruitment of vascular smooth muscle cells (VSMC: 0 vs 0 vs 2.2±0.9%, P<0.001) to the neovasculature. These data indicate that VEGFR1/VEGFR2/PDGFR‐β/cKit signalling is required for eNOS stimulated angiogenesis, and for the first time demonstrate increased vessel diameter and VSMC recruitment following PTK787 treatment.

All data presented eGFP vs. eNOS vs. eNOS+PTK787; mean±SEM; n=6. Supported by BHF.
Kidney Injury Response to Elevated Blood Pressure vs Increased Intrarenal Ang II in 2K1C Goldblatt Hypertensive Rats
Authors: Carter Brandon J , Martin Victoria L , Navar L Gabriel , Prieto Minolfa C
Published: June 2021

This study determined the relative role of elevated blood pressure (BP) versus increased Angiotensin II (Ang II) content on the tissue injury response in both kidneys (clipped ‐CK‐ and nonclipped ‐NCK) of two kidney‐one clip (2K1C) Goldblatt hypertensive rats. Kidney injury markers of arterial wall proliferation (α‐sma), fibrosis (Collagen III), inflammation (CD68), cell proliferation‐regeneration (PCNA, Vimentin), and hypoxia (HIF‐α1) were examined using immunohistochemistry on paraffin embedded kidney sections from 2K1C male Sprague‐Dawley (n=6) rats and sham (n=4) rats. After 25 days of unilateral renal clipping (0.25mm), systolic BP increased in 2K1C rats compared to sham rats (184±9 vs.121±6 mmHg). Semi‐quantitation of injury markers showed increased afferent arteriole wall proliferation, macrophages number, and interstitial expansion in both kidneys of 2K1C rats. However, tubular cell proliferation and regeneration were only augmented in the NCK of 2K1C rats compared to CK and sham kidneys. Increased intrarenal Ang II content in both kidneys of 2K1C rats contributes to renal fibrosis and inflammation in Goldblatt hypertension, but preferentially increases of epithelial‐to‐mesenchymal transition and tubule‐cell proliferation in the NCK seems to be associated to the concurrent effect of elevated BP and high intrarenal Ang II content.

Support from the NIH and LAMP‐Tulane University.
Potential Effect of β‐Estradiol in Human Jurkat T‐Cells
Authors: Hullitt JoAnna , Davis Corey , Yedjou Clement , Cameron Joseph A
Published: June 2021

β‐estradiol is the most potent estrogen of a group of endogenous estrogen steroids which includes estrone and estriol. This steroid hormone is the most potent natural estrogen, produced mainly by the ovary, placenta, and in smaller amounts by the adrenal cortex, and the male testes. Although β‐estradiol protects the renal and cardiovascular systems, the mechanisms involved remain unclear. In this research, we performed both MTT assay and trypan blue exclusion test to: 1. evaluate the effect of β‐estradiol in HL‐60 and Jurkat T‐cells and 2. compare the sensitivity of these two cells types. The results from both MTT assay and trypan blue exclusion test demonstrated that low, physiological levels of β‐estradiol induce cellular proliferation in Jurkat T‐cells. At higher dose of exposure (16 uM), β‐estradiol decreases the viability of Jurkat T‐cells compared to the control cells. A similar trend was obtained with the trypan blue exclusion test using the hemacytometer to count the cells manually. In summary, the results of the present study demonstrate that physiological levels of β‐estradiol induce cell growth, and cellular proliferation of Jurkat T‐cells, whereas higher doses inhibit cell growth and induce cell death. Supported in part by NIGMS‐NIH Grant R25 GM50117 and RCMI‐NIH Grant G12RR13459.
Cigarette smoke exposure dose‐dependently alters the activity and coupling of endothelial nitric oxide synthase in the endothelium.
Authors: Wang Tse‐Yao , Druhan Lawrence J , Chen Chun‐An , Zweier Jay L
Published: June 2021

A critical mechanism implicated in cigarette smoke (CS) induced cardiovascular diseases is the inhibition of endothelial nitric oxide synthase (eNOS). Here we show that CS extract (CSE) alters endothelial function by an eNOS‐dependent mechanism. We demonstrate that treatment with CSE impairs vessel relaxation; we show that NO generation from endothelial cells, as measured by EPR spin‐trapping, was decreased by 36%, 43%, or 48% with 10%, 20%, or 50% CSE treatment, respectively. Subsequent addition of tetrahydrobiopterin (BH4) restored eNOS activity in cells treated with >50% CSE, however the decrease in eNOS activity produced by higher doses of CSE produce was irreversible. The addition of BH4 to cells treated with >20% CSE actually led to an increase in eNOS activity. We found no change in eNOS protein levels in cells treated with low concentrations of CSE, while there was a significant decrease in eNOS protein following treatment with CSE concentrations >50%. Additionally, we found that Thr495, phosphorylation of which has been reported to negatively regulate eNOS, is time dependently dephosphorylated by treatment with CSE. Thus, low level CSE treatment leads to reversible inhibition of eNOS via oxidation of BH4, and exacerbates the potential eNOS‐dependent oxidative stress via dephosphorylation of Thr495. Conversely, high CSE concentrations irreversibly inhibit via a reduction in total eNOS protein.
Effects of Arginase I and II on the local production of NO
Authors: Elms Shawn Christopher , Morris Sidney , Caldwell William , Fulton David
Published: June 2021

Endothelial Nitric Oxide Synthase (eNOS) produces nitric oxide (NO) within the endothelial cell layer of blood vessels. NO is a potent vasodilator and perturbations of NO signaling have been associated with cardiovascular diseases such as diabetes, atherosclerosis and hypertension. L‐arginine is a semi‐essential amino acid that is the substrate for eNOS. However, L‐arginine is also the substrate for the arginases, enzymes that metabolize L‐arginine into ornithine and urea. The potential for arginases to compete with eNOS for arginine has been investigated as an underlying reason for endothelial dysfunction and a mechanism to account for the arginine paradox. Previously we showed that the activity of eNOS constructs targeted to different intracellular locations was equally inhibited by Arginases I and II, despite their different locations within the subcellular environment. These results argue against the concept that spatially restricted pools of L‐arginine account for the arginine paradox. Furthermore, the addition of excess extracellular L‐arginine failed to override the inhibitory effect of arginase I which suggests a more complex mode of eNOS inhibition. Therefore the current study was designed to investigate the involvement of other mechanisms by which arginases inhibit eNOS and included cell free activity assays, inhibition of arginases, eNOS monomer/dimer ratios and superoxide production.