bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of G‐protein‐coupled estrogen receptor (GPER) inhibits coronary artery smooth muscle cell proliferation
Authors: Li Fen , Wang Gang , Zhou Beiyan , White Richard , Heaps Cristine , Stallone John , Han Guichun
Published: June 2021

One aspect of the beneficial influences of 17β‐estradiol on vascular smooth muscle cells (VSMC) is inhibition of VSMC proliferation following vascular injury. GPER is a potential mediator of estrogen action on coronary arteries, but whether GPER plays a role in coronary artery smooth muscle proliferation has never been reported. The objective of our study was to test the functional role of GPER in human and porcine coronary (CASMC) proliferation by applying flow cytometric analysis and Western blotting. The results are as follows: G‐1 (GPER agonist) inhibited both human and porcine CASMC proliferation in a concentration and time‐dependent manner (10−8 to 10−5 M). Flow cytometry revealed that G‐1 significantly decreased the proportion of S‐phase and G2/M cells in the growing cell population (stimulated by 10% charcoal/dextron striped FBS or 10 nM PDGF‐BB), suggesting that G‐1‐induces cell proliferation by slowing down the progression of the cell cycle. Western blot revealed that G‐1‐induced cell cycle retardation was associated with decreased expression of cyclinB, up‐regulation of cyclinD1, and concomitant induction of p21. G‐1 treatment also inhibited the phosphorylation of ERK1/2 and Akt. We conclude that GPER activation inhibits CASMC proliferation by retardation of cell cycle progression via inhibiting ERK1/2 and Akt activities.
Doxycycline protects against right ventricular dysfunction and dilatation caused by acute pulmonary thromboembolism
Authors: Neves Evandro Manoel Neto , Santos Ozelia Sousa , Ferraz Karina Coutinho , Ceron Carla Speroni , Romano Minna Dias , Gali Luis Gustavo , Maciel Benedito Carlos , Santos Jose Eduardo Tanus
Published: June 2021

We examined whether pretreatment with doxycycline (a matrix metalloproteinase MMP inhibitor) protects against acute pulmonary thromboembolism (APT)‐induced right ventricular dysfunction and dilatation.

APT (350 mg/kg of autologous blood clots) increased mean pulmonary artery pressure (MPAP) and pulmonary vascular resistance index (PVRI) by approximately 186% and 196% respectively. Ecocardiography showed that right ventricle (RV) diameter increased in animals with APT (from 10.7±0.8 to 18.3±1.6 mm, P<0.05), but not in embolized animals pretreated with doxycycline at 10 mg/kg i.v. (from 13.3±0.9 to 14.4±1.0 mm, P>0.05). Dobutamine stimulation induced minor improvements in RV function in embolized animals. However, doxycycline improved the RV contractile reserve. APT increased serum cardiac troponin I concentrations and RV gelatinolytic activity, and doxycycline prevented theses increases (P<0.05).

These results suggest that MMPs are involved in APT‐induced cardiomyocyte injury, RV dysfunction and dilatation, and degrade contractile proteins. Doxycycline improves the responses to dobutamine during APT.

Funded by FAPESP, CNPq, and CAPES (Brazil).
G protein‐coupled receptor (GPCR) arrays identify physiologically relevant targets in Pulmonary Artery Smooth Muscle Cells (PASMC): mRNA to Function
Authors: McDonald Daniel Scott , Aroonsakool Nakon , Kwon Ohmin , Insel Paul A , Murray Fiona
Published: June 2021

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance, in part due to increased pulmonary artery smooth muscle cells (PASMC). Since cyclic AMP (cAMP) decreases proliferation of PASMC, G protein‐coupled receptors (GPCRs) that couple Gαs are potential targets for PAH. Using a TaqMan®GPCR array and human PASMC we identified that PASMC express >130 GPCRs, at least 50 of which regulate cAMP levels. Using real‐time PCR and GPCR agonists, we investigated the relationship between GPCR expression and function by testing if the highest, intermediate and lowest expressed Gαs‐coupled GPCRs (determined by cycle threshold [ΔCt]) in PASMC generate relative amounts of cAMP (by radioimmunoassay). The highest, intermediate and lowest Gαs‐coupled GPCR that we tested were the adenosine 2B receptor (A2BR, ΔCt = 18), the prostaglandin E receptor 2 receptor (PGE2R, ΔCT = 22) and the gastric inhibitory polypeptide receptor (GIPR, ΔCt = 24). cAMP accumulation (fmol) in response to receptor agonists, CV1808 (A2BR, 1μM, 1.8 fmol), Butaprost (PGE2R, 1μM, 1.2 fmol) and GIP (GIPR,1μM, 0.7 fmol) correlated with receptor mRNA expression (r2=0.90). Moreover, cAMP dose‐dependently decreased PASMC proliferation. Our data show that the mRNA expression of Gαs‐coupled GPCRs predicts function, implying such an approach can identify GPCRs in PASMC that may be therapeutic targets for PAH. NIH funded.
A major constituent from Zanthoxylum nitidum, compound X, is a naturally occurring antagonist of the human and rat pregnane X receptor
Authors: Ma Chia-Yun , Lim Yun-Ping
Published: June 2021

Discrepancy expression and regulation of drug‐metabolizing enzymes (DMEs) is a common cause of adverse drug effects in some drugs with narrow therapeutic index (TI). A important cytochrome P450 3A4 (CYP3A4) is predominantly regulated by a nuclear receptor, pregnane X receptor (PXR). A pure compound isolated from Zanthoxylum nitidum, compound X, exhibits variety of biological functions, however the effect of compound X on the modulation of CYP3A4 is not well understood. In this study, the effects of compound X on the PXR‐CYP3A4 pathway, and the underlying mechanisms, were characterized. Compound X potently and dose‐dependently attenuated CYP3A4 induction by blocking the activation of nuclear receptors, especially PXR. Further mechanistic studies revealed that compound X inhibited PXR by interrupting the binding of steroid receptor cofactor‐1 (SRC‐1) and hepatocyte nuclear factor 4α (HNF4α). Our results may lead to the development of important new therapeutic and dietary approaches to reduce the frequency of undesirable drug interactions. Here, we established compound X as a novel and natural potent inhibitor of PXR and can be a useful tools for modulating DME expression and drug efficacies. Modification of CYP3A4 expression and activity by consumption of compound X could have important implications for drug safety.
Impact of the EphrinB/EphB system on pro‐inflammatory monocyte‐endothelial cell interaction
Authors: Liu Hui , Braun Jennifer , Hecker Markus , Korff Thomas
Published: June 2021

The orchestrated expression of ephrinB ligands and EphB receptors is a prerequisite for the development of the vasculature. However, only little is known about the function of this ligand/receptor system in the adult vasculature. Our results show that ephrinB2 as well as ephrinB1 are localized on the luminal surface of endothelial cells and up‐regulated during inflammation, enabling their interaction with circulating leukocytes. Thus, ephrinB1/2 expression on endothelial cells promotes the adhesion and transmigration of EphB2‐expressing monocytes. Whereas forward signaling downstream of the EphB receptors promotes the migratory and pro‐inflammatory activity of monocytes, reverse signaling induces a JNK‐dependent increase in expression of adhesion molecules such as VCAM‐1 or E‐selectin in the endothelial cells. In order to analyse these ephrinB/EphB‐dependent interactions between monocytes and endothelial cells in greater detail, we generated EphB2‐overexpressing mouse myeloma cells, which in fact are capable of upregulating adhesion molecule expression and von Willebrand factor release in the cultured endothelial cells. We conclude that reverse signaling processes promote the pro‐inflammatory differentiation of endothelial cells whereas forward signaling facilitates monocyte diapedesis and subsequent differentiation into macrophages in the vessel wall.
Syndecan‐1 modulates leukocyte adhesion in the murine parietal peritoneum microcirculation in response to Staphylococcus aureus lipotechoic acid
Authors: Kowalewska Paulina , Fox-Robichaud Alison E.
Published: June 2021

Syndecan‐1 modulates leukocyte recruitment in several tissues. The objective of this study was to determine the effects of syndecan‐1 blockade on leukocyte‐endothelial cell interactions in venules of the parietal peritoneum. BALB/c mice were stimulated with ip injection of E. coli LPS or S. aureus LTA and the microcirculation was examined by intravital microscopy after 4 hrs. Syndecan‐1 was localized to the basal membrane of venular endothelium using in vivo confocal imaging with a fluorescent anti‐syndecan‐1 antibody. Leukocyte‐endothelial interactions and leukocyte extravasation were not significantly different in response to LPS compared to naive control. After stimulation with LTA, rolling (94.8 ± 15.3 cells/min) and adhesion (15.0 ± 4.0 cells) along the endothelial wall and extravascular leukocytes (8.5 ±1.8 cells) were significantly increased compared to control, (37.0 ± 8.5, 1.26 ± 0.5, 0.7 ± 0.3 respectively, p < 0.05). Syndecan‐1 blockade before stimulation with LTA significantly increased the number of adherent leukocytes (37.8 ± 6.0, p < 0.001) and systemic neutrophil counts. Leukocyte‐endothelial interactions and systemic neutrophil counts were not increased in anti‐syndecan‐1‐injected saline controls. Our data suggest the parietal peritoneum vasculature is more responsive to LTA than LPS and syndecan‐1 is a negative regulator of leukocyte adhesion during LTA‐induced inflammation.
Atherosclerosis development is differentially regulated in whole genome vs. bone marrow‐derived haploinsufficient NFAT5/ApoE null mice
Authors: Halterman Julia Ann , Kwon H Moo , Wamhoff Brian R
Published: June 2021

We have previously shown that the transcription factor, nuclear factor of activated T‐cells 5 (NFAT5), plays a role in vascular smooth muscle cell phenotypic modulation in acute injury, but the role of NFAT5 in atherosclerosis is unknown. Our main objectives were to determine if genome‐wide NFAT5 haploinsufficency (NFAT5+/−) altered atherosclerotic development and to determine whether NFAT5 expression in bone marrow (BM)‐derived cells affected lesion progression. NFAT5+/−ApoE−/− mice were placed on a high fat diet for 20 wks and analysis of the thoracic aorta and aortic arch established that genome‐wide NFAT5 haploinsufficiency reduced atherosclerotic lesion formation by 73% and 43%, respectively, but did not alter lesion size in the aortic root. BM transplant studies revealed that transplantation of NFAT5+/−ApoE−/− BM into NFAT5+/+ApoE−/− mice resulted in a similar 86% reduction in lesion formation of the thoracic aorta, yet produced an unexpected 37% increase in total lesion area of the aortic root. Immunohistochemical analysis of root lesions revealed that mice transplanted with NFAT5‐deficient BM had increased macrophage investment of the lesion. We have identified that NFAT5 expression in BM‐derived cells is critical for the regulation of lesion morphometry and composition, and we show that NFAT5 deficiency in macrophages leads to their increased investment of aortic root lesions.
Extravascular platelets in corneal microvascular inflammation: a potential determinant of recovery from wound injury
Authors: Rumbaut Rolando , Li Zhijie , Burns Alan R , Smith C Wayne
Published: June 2021

The notion that platelets extravasate across inflamed microvessels is not well appreciated and their functional role is unknown. Recently, we reported evidence of extravascular platelets surrounding inflamed venules in a mouse model of corneal wound injury, and of a functional role for platelets in nerve recovery in this model. Whether extravascular platelets are required for nerve recovery remains unclear. In this study we assessed the relationship between microvascular platelet recruitment (intra vs. extravascular, at 12 h) and nerve recovery (at 96 h) following wound injury. We studied wild type (WT) mice, and mice deficient in either ICAM‐1 or gamma‐delta T cells. Nerve recovery was reduced by ~50% in both strains of deficient mice. Interestingly, the number of intravascular platelets was comparable among all strains, while the number of extravascular platelets was markedly reduced (by > 85%) in mice deficient in ICAM‐1 and gamma‐delta T cells. These data suggest a functional role for extravascular platelets in nerve recovery following corneal injury.

Support: NIH EY018239, EY17120, HL079368
Effects of SOD/PEG and soy flour on insulin sensitivity of blood platelets of atherogenic dieted rabbits
Authors: Beard Elizabeth Letitia , Mena Juan Carlos , Van Blaricom Amy
Published: June 2021

The effects of antioxidant superoxide dismutase (SOD) injections and of SOD rich soybean flour were studied in New Zealand strain, male rabbits. The animals were fed an atherogenic diet (AD) of 2% Cholesterol, 6% Corn Oil and 92% Purina Rabbit Chow, for 5 weeks prior to starting the experiments using 3 experimental groups of 5 animals. Group I AD Controls –theADC group‐fed AD, Group II fed AD and injected with 2,000units SOD‐PEG −2 x/week‐IP‐ (approximately 11,000 units/gm hemoglobin) ‐ the AD‐SOD group, and Group III was fed AD with 5% SOD rich soybean flour added‐the SF+AD group. The treatments lasted for 5 additional weeks before the animals were sacrificed. Sensitivity to insulin was measured in blood platelets in each group by assaying their glucose uptake following treatment with insulin. Blood plasma and platelets were also assayed for cholesterol and phospholipid. levels. Mean glucose assays of insulin treated platelets from AD‐SOD rabbits measured 8.2 mg/dl of glucose vs 4.5 mg/dl from ADC .platelets‐an 82.2% increase. SF+AD platelets registered a 10.1% increase in insulin sensitivity vs ADC samples. Mean cholesterol levels ( mg/dl ) in blood platelets diminished by 18.8% in the AD‐SOD group and 10.7% in the SF+AD platelets compared to the ADC values. Mean plasma cholesterol levels fell by 16.8% in the AD‐SOD and 19.1% in SF+AD vs ADC plasma. Mean phospholipid levels (mg%) showed a decrease of 12.5% in AD‐SOD platelets and 9.2% in SF+AD platelets vs those in ADC assays. Mean phospholipid levels also fell in AD‐SOD by 17.3% and by 15.8% in SF+AD vs the ADC control values.
Disruption of Nrf2 signaling impairs angiogenic capacity of endothelial cells: implications for microvascular aging
Authors: Ungvari Zoltan , Valcarcel-Ares M. Noa , Gautam Tripti , Warrington Junie P. , Bailey-Downs Lora C. , Sosnowska Danuta , de Cabo Rafael , Sonntag William E. , Csiszar Anna
Published: June 2021

The redox‐sensitive transcription factor Nrf2 plays a key role in preserving a healthy endothelial phenotype and maintaining the functional integrity of the vasculature. Previous studies demonstrated that aging is associated with Nrf2 dysfunction in endothelial cells, which alters redox signaling and likely promotes the development of large vessel disease. Much less is known about the consequences of Nrf2 dysfunction at the level of the microcirculation. To test the hypothesis that Nrf2 regulates angiogenic capacity of endothelial cells, we determined whether disruption of Nrf2 signaling (by siRNA knockdown of Nrf2 and over‐expression of Keap‐1, the cytosolic repressor of Nrf2) impairs angiogenic processes in cultured human coronary arterial endothelial cells (CAECs) stimulated with VEGF and IGF‐1. In the absence of functional Nrf2 CAECs exhibited impaired proliferation and adhesion to vitronectin and collagen. Disruption of Nrf2 signaling also reduced cellular migration (measured by a wound‐healing assay using electric cell‐substrate impedance sensing [ECIS] technology) and impaired the ability of CAECs to form vascular‐like structures. Collectively, we find that Nrf2 is essential for normal endothelial angiogenic processes, suggesting that Nrf2 dysfunction may be a potential mechanism underlying impaired angiogenesis and microvascular rarefaction in aging.
Effect of chronic hypertension on cortical parenchymal arteriole tone and K+ mediated vasodilation
Authors: Iddings Jennifer , Filosa Jessica Andrea
Published: June 2021

Hypertension blunts functional hyperemia, a response in which increased neuronal activity causes local increases in cerebral blood flow. The mechanism mediating this occurrence is unknown. To test the hypothesis that increased vascular reactivity and altered vascular smooth muscle cell (VSMC) Ca2+ dynamics are involved in the blunted hyperemic response, we measured vascular responses and VSMC Ca2+in cortical parenchymal arterioles from normotensive (WKY) and hypertensive (SHR) rats. In the presence of the thromboxane agonist U46619 (150 nM), baseline tone and responses to the potent vasodilatory signal K+ (10 mM) were significantly higher in SHR vs. WKY (42.06% vs. 29.78%; P=0.0173 and 26.63% vs. 12.43%; P=0.0133 respectively). VSMC Ca2+ oscillation frequency induced by U46619 was significantly higher in SHR vs. WKY (0.1999Hz vs. 0.1493Hz; P=0.0007) whereas VSMC Ca2+ amplitude was not changed between groups. To conclude, both vascular tone and VSMC Ca2+ oscillation frequency are increased in SHR indicating that alterations in VSMC Ca2+dynamics likely contribute to increased parenchymal arteriole tone observed during hypertension. Further, responses to extracellular K+ are increased in SHR supporting previous data from our lab which demonstrates that the magnitude of dilatory responses is dependent on the degree of steady‐state baseline tone. AHA 11PRE7400037 to JAI; NIH 1R01HL089067‐01A2 to JAF.
Beneficial effects of melatonin combined with exercise on endogenous neural stem/progenitor cells regeneration after spinal cord injury
Authors: Lee Youngjeon , Park Kanghui , Lee Sang-Rae , Chang Kyu-Tae , Hong Yonggeun
Published: June 2021

After spinal cord injury (SCI), as a protective response, endogenous adult neural stem/progenitor cells (NSPCs) proliferate and differentiate into neurons and glial cells to facilitate healing process. We have shown that melatonin treatment combined with exercise had a synergistic effect in functional recovery after SCI. In this study, we have examined the combined effects of melatonin and exercise (SCI+MT+Ex) on NSPCs, at early and late phases of spinal injury. Compared to the SCI group, there was marked improvement in hind limb function in SCI+MT+Ex group after 14 and 21, as documented by the reduced size of the spinal lesion and a higher density of dendritic spines and axons; such functional improvements were associated with increased numbers of BrdU‐positive progenitor cells. Furthermore, neuronal marker MAP‐2 was increased in the injured thoracic segment, while astrocyte marker GFAP was increased in the cervical segment, along with elevated numbers of BrdU‐positive nestin expressing neural precursor cells in the SCI+MT+Ex group. These results suggest that the synergistic effect of melatonin combined with exercise might create a microenvironment to facilitate proliferation of nestin‐positive endogenous NSPCs to effectively replace injured cells and to improve regeneration of SCI.
Cerebral Compliance Changes With Sympathetic Activation During Cold Pressor Test
Authors: Witkowski Jaclyn , Falvo Michael , Blatt Melissa , Jasien Jessica , Deegan Brian M , OLaighin Gearoid , Serrador Jorge M
Published: June 2021

The role of sympathetic activation on arterial compliance has yet to be examined. This study examined cerebral flow velocity and cerebral compliance in response to sympathetic activation.

Healthy subjects (n=13) performed a cold pressor test in which their forearms were immersed in a bucket of ice water for up to 3 minutes. Cerebral flow velocity (CFV) (middle (MCA), anterior cerebral (ACA), vertebral arteries (VA), external carotid), blood pressure, and end‐tidal CO2 were measured. Data was collected at baseline, first and final 15 sec of immersion.

Compared to initial immersion, CFV increased the most in the last 15 sec of the protocol: ACA (+9.5±3.2%, P<0.02), MCA (+10.0±2.8%, P<0.02), VA (+15.4±4.6%, P<0.02). At the end of the protocol, significant increases in blood pressure were seen (+15.5±2.5 mmHg, P<0.001). Pulse wave velocity (PWV) increased in the periphery from baseline and through each time point: ACA (7.2±0.4 to 8.5±0.8 to 8.5±0.7 m/s); MCA (7.2±0.4 to 9.9±1.6 to 9.2±0.6 m/s); VA (6.7±0.5 to 7.0±0.4 to 8.5±1.4 m/s).

Findings suggest that increasing sympathetic activity does not cause cerebral vasoconstriction, but increases CFV while reducing arterial compliance. Sympathetically mediated reductions in compliance may be an adaptive mechanism to enhance pressure transmission to the brain when humans are upright and pressure at the brain is lower due to the hydrostatic gradient. Supported by NASA/NIH.
Effects of acute moderate‐ and high‐intensity exercise on glucose disposal and beta‐cell function
Authors: Rynders Corey A , Chan Alice , Weltman Judy Y , Barrett Eugene J , Weltman Arthur
Published: June 2021

The effects of isoenergetic high (HIE) and moderate intensity (MIE) cycle ergometer exercise on glucose disposal and beta‐cell function were examined in nine sedentary pre‐diabetic adults. Subjects completed 3 conditions with order randomized: HIE (84% VO2peak), MIE (51% VO2peak), and seated rest (C). One‐hour post‐exercise (or C), subjects received a 3‐hr OGTT. Plasma glucose, insulin, and C‐peptide concentrations were measured before and at 5–10min intervals after glucose ingestion. The incremental area under the glucose curve was calculated for the 0–60min (AUC‐1), 60–120min (AUC‐2), and 120–180min (AUC‐3) time intervals. Deconvolution analysis of C‐peptide quantified insulin secretion rate. Beta‐cell function was assessed using the insulin secretion/insulin resistance index. Compared to C, HIE decreased the mean glucose AUC‐2 by 36% (p=0.03) and AUC‐3 by 42% (p=0.003) whereas MIE tended to reduce AUC‐3 by 21% (p=0.06). Glucose AUC‐2 was 27% lower after HIE compared to MIE (p=0.008). A trend for improved beta‐cell function was observed after HIE (C=0.34±0.05; MIE=0.36±0.16; HIE=0.42±0.17; p=0.17, C vs. HIE). These results suggest that exercise has an intensity dependent effect to improve postprandial glycemic excursion which may in part be related to acute enhancements in beta‐cell function. This research was supported by grant funding from Virginia's Commonwealth Health Research Board.
Nebivolol induced vasodilation of renal afferent arterioles involves β3 adrenergic receptor activation
Authors: Feng Ming-Guo , Navar L. Gabriel
Published: June 2021

Nebivolol (Neb) is a novel β1 adrenergic blocker that also elicits vasodilation. However, the renal vasodilator mechanisms remain unclear. Videomicroscopic measurements of vascular dimensions were performed on rat isolated blood perfused juxtamedullary nephrons to test the hypothesis that Neb induced vasodilation of afferent arterioles (AA) involves β3 receptor activation stimulating NOS and NO. Single AA were superfused with Neb either alone or combined with selective β adrenergic blockers: metoprolol (MTP, β1), butoximine (BTX, β2), SR59230A (SR, β3); the sGC inhibitor, ODQ and the NOS inhibitor, L‐NNA. Neb 100μM increased AA diameters by 17.4±2.3% (n=12, p<0.01); however, MTP up to 80μM did not elicit significant changes in AA diameters (1.3±0.7%, n=6) and did not prevent the effects of Neb to dilate AA diameters (15.0±2.5%, n=6, p<0.01). Likewise, BTX failed to block the Neb effect. In contrast, the Neb induced vasodilation was markedly attenuated by SR 5μM (4.9±1.1% vs baseline, n=6, p>0.05). Pretreatment with ODQ 10μM (n=5) or L‐NNA 100μM (n=6) prevented the vasodilation response to Neb. These data indicate that the vasodilator effect of Neb is via a mechanism that is independent of β1 receptors but is predominantly mediated via NOS‐NO‐sGC‐cGMP dependent mechanisms initiated by activation of β3 receptors presumably on AA endothelial cells. (Supported by grant BYSMD62 from Forest Research Institute US)
Role of Adenosine A1 Receptors in Regulation of Arteriolar Responses to Adenosine and Angiotensin II
Authors: Gao Xiang , Carlström Mattias , Fredholm Bertil , Persson Erik
Published: June 2021

OBJECTIVE

Adenosine (Ado) mediates tubuloglomerular feedback, whereas AngII and NO are important modulators. A1‐deficiency abolish TGF and diminish effectiveness of L‐NAME and AngII to constrict renal resistance vessels. We used A1‐knockout (A1−/−) and wild‐type (A1+/+) mice to investigate the synergism between AngII and low‐dose Ado in regulation of arteriolar responses.

METHODS

Contractions were measured in renal afferent arterioles from nontreated mice and those with 14‐days pretreatment with L‐NAME (10‐4M) or AngII (400 ng/kg/min). Concentration response curves were obtained by Ado (10‐8M; 15 min) alone, or in combination with cumulative application of AngII (10–12 to 10‐6M).

RESULTS

Ado contracted arterioles from A1+/+ (11%), but had no significant effect in A1−/−. Ado significantly enhanced AngII‐mediated contraction in both genotypes, however, the contractile response was stronger in A1+/+ (56%) than in A1−/− (40%). Prolonged treatment with L‐NAME or AngII did not change the responses to Ado alone. However, arteriolar response to combination with Ado+AngII was enhanced in A1+/+, but was attenuated in A1−/−.

CONCLUSION

Low‐dose Ado augments AngII‐induced constriction in both A1+/+ and A1−/−, by non‐receptor‐mediated actions. Underlying mechanisms may involve modulation of NO bioavailability or intracellular effects of Ado, introduced by Ado‐transporters.
Glyceollins induce cytoprotective effect by enhancing glutathione synthesis through Nrf2 in hepatoma cells
Authors: Kim Jong-Sang , Kim Hyo Jung , Jung Chaelim , Park In-Sil , Lim Ji Sun
Published: June 2021

The purpose of this study was to investigate the protective effects of glyceollins against excessive oxidative stress induced by GSH depletion. In mouse hepatoma cells (Hepa1c1c7) subjected to the buthionine sulfoximine (BSO), an inhibitor of gamma‐glutamylcysteine synthetase, the intracellular GSH content was significantly lowered. On the other hand, the incubation with glyceollins in the presence of BSO increased the level of GSH, expression of GSH synthesis enzyme, and nuclear translocation of NF‐E2‐related factor‐2 (Nrf2), compared to the cells treated with BSO only. Nrf2‐antioxidant responsive element (ARE)‐reporter activity assay in HepG2‐C8 showed that BSO increased the ARE‐reporter activity in a dose dependent manner, compared to vehicle‐treated cells, while the cotreatment with glyceollins caused further increase in reporter luciferase activity than BSO alone. Taken together, these results suggest that glyceollins exhibit cytoprotective effects in BSO‐treated cells from oxidative stress by further inducing glutathione synthesis, which was mediated through Nrf2 activation.
Inner Gate Residues Contribute To Sugar and Na+ Binding in SGLT1
Authors: Jiang Xuan , Loo Donald D.F. , Hirayama Bruce A. , Wright Ernest M.
Published: June 2021

Human SGLT1 belongs to a class of membrane transporters that convert the energy of Na+ electrochemical gradients to drive uphill transport of sugar. Crystal structures demonstrate that the sugar‐binding site is sandwiched between hydrophobic intra‐ and extracellular gates. A tyrosine forms the intracellular gate, and appears to stack with the pyranose ring of glucose. This tyrosine residue, along with the two flanking residues, constitutes an aromatic motif that is conserved in the SGLT gene family. To investigate their function we constructed hSGLT1 mutants (W289, Y290 and W291), expressed them in oocytes, and measured their steady state and pre‐steady state transport kinetics, using biophysical and biochemical assays. No functional expression was obtained for W289 mutants. Substitution of Y290 and W291 with cysteine led to the dramatic reduction in apparent sugar binding affinity (K0.5 > 100 mM), and the affinity was largely restored for the phenylalanine mutants (Y290F and W291F). This confirms that the interaction of sugar with these aromatic side chains is important for sugar transport. Substitution of residues Y290 and W291 also decreased Na+ binding affinity in the presence or absence of sugar, and lowered the Na+: glucose coupling ratio from 2 to 1, suggesting that these residues are also important in Na* coordination in the Na1 binding site. Supported by NIH Grant DK19571 to E.M.W.
Unraveling ammonia transport: Role of NHE2/3 and cH/K‐ATPase
Authors: Engevik Melinda Anne , Worrell Roger T , Shull Gary E
Published: June 2021

In liver failure, hepatic ammonia clearance is impaired; this can result in hyperammonia and hepatic encephalopathy (HE). Current therapy for HE targets colonic microflora to reduce ammonia production and thus delivered liver load. A better understanding of colonic ammonia transport mechanisms is necessary to more directly target epithelial transport. Ammonia secretion occurs in colonic epithelial cells via basolateral NH4+ uptake mediated by NKCC1 and Rhesus glycoprotein B (RhBG). The apical ammonia exit pathway likely occurs via ion trapping involving the combined action of Rhesus glycoprotein C (RhCG) and H+ transport via cH/K‐ATPase and NHE2/3. Quantitative PCR (qRT‐PCR) was used to define distal colon transporter expression in various transporter null mice to establish co‐operative function in ammonia secretion. NKCC1 and RhCG mRNA was significantly higher in distal colon of cH/KATPase−/− mice verses WT littermates. NKCC1 and RhBG mRNA were significantly decreased in distal colon of NHE3−/−mice verses WT littermates whereas no difference was observed in NHE2−/− mice. In addition, cH/KATPase mRNA was increased in NHE3−/− proximal and distal colon. In the human colonic cell line T84, inhibition of NHE2/3 by amiloride resulted in decreased secretory NH4+ flux. These data suggest that NHE3 is the principle pathway for H+ exit allowing for luminal NH3 ion trapping. Supported by NIH DK079979.
Structural determination of OATP transporters utilising homology models and cell based assays
Authors: Taylor-Wells Jennina C. , Meredith David , Kelly Sarah
Published: June 2021

Membrane transport proteins (transporters) are present in the lipid bilayer of cells and selectively transport compounds across the membrane. The organic anion transporting polypeptides (OATPs) are a family of membrane transporters expressed in a variety of tissues, including absorptive/excretory cells of the liver, kidney and intestine. In addition, OATPs transport an array of endogenous and xenobiotic compounds including bile salts, thyroid hormones, statins and antineoplastics. OATPs play an important role in cell homeostasis and drug absorption/excretion, and have also been implicated in disease and drug‐drug interactions. However there is still much to determine regarding the binding properties and exact structure of these proteins. The topology and binding properties of OATP1A2, OATP1B1, OATP1B3 and OATP2B1 have been investigated using a HEK293T cell model.

OATP proteins have been expressed in HEK293T cells and kinetic properties determined using the OATP substrate estrone‐3‐sulfate. Bioinformatics analysis using various topology models has revealed that OATP1B1, OATP1B3 and OATP2B1 have between 11–12 transmembrane domains. However there could be as few as 8 domains, as is the predicted topology of the less well characterised OATP1A2. The topology of these proteins has been experimentally investigated using a FLAG epitope tag recognition system. Epitope tags were cloned into the putative extracellular regions of the protein, which were recognised by a FLAG antibody. The detection of the antibody was quantified by a chemiluminescence method and those detected confirmed as extracellular regions. The findings of this study will be used to further determine drug transport pathways and aid in future drug development.
Mechanosensing by KATP channels and PECAM‐1 contributes to superoxide generation in mouse model of lung ischemia
Authors: Noel John , Hong Nankang , DeBolt Kris , Fisher Aron B. , Chatterjee Shampa
Published: June 2021

Vascular responses to shear stress arising from blood flow entail the involvement of mechanosensors on endothelial cells. We showed earlier that the KATP channel is an important component of the flow sensing machinery in the pulmonary endothelium. Based on previous studies (E. Tzima, Nature, 2005), we posited that the platelet endothelial cell adhesion molecule 1 (PECAM‐1), by virtue of its junctional location and cytoskeletal linkage, could serve as a mechanosensor that senses loss of blood flow. Using an isolated perfused mouse lung and confocal imaging of ROS probes in the pulmonary microvasculature, we detected ROS generation upon loss of shear (ischemia) in wild type lungs that was significantly attenuated both in KATP and PECAM‐1 knockout (KO) mouse lung. By image analysis, KATP KO and PECAM‐1 KO were capable of generating only 62% and 51% of the ROS level detected in wild type (WT), respectively. High‐performance liquid chromatography (HPLC) of dihydroethidium extracted from the ischemic lung was used to detect superoxide (SO). In agreement with the imaging data, HPLC analysis showed a partial attenuation of SO in the PECAM‐1 KO, indicating SO as the parent oxidant species and reinforcing the confocal imaging method. These results indicate that KATP channel and PECAM‐1 may be a part of a mechanosensing machinery that contributes to the generation of SO in a shear‐dependent manner. NHLBI‐T32‐S54932
Antenatal nicotine exposure results in programming of aberrant alveolar development and interstitial pulmonary fibrosis in adult male rats
Authors: DASGUPTA CHIRANJIB , Xiao DaLiao , Xu Zhice , Yang Shumei , Zhang Lubo
Published: June 2021

Objective

Identification of abnormalities in lung structure, and expression of profibrotic proteins in the adult rat offspring lung exposed to maternal smoking in fetal life.

Methods

Antenatal nicotine exposed adult offspring lungs were analyzed for lung volume (LV), lung/body weight ratio (L/BWR), morphology, and protein expression patterns.

Results

Male offspring lung showed decreased radial alveolar count, thickened alveolar septa, with heightened interstitial collagen expression, angiotensin II type 1 receptor (AT1R), TGF‐ β1, and CTGF proteins. In contrast, female offspring lungs had reduced L/BWR, increased LV, and expression of angiotensin II type 2 receptor (AT2R), resulting in the decreased AT1R to AT2R ratio. Smad3 expression was down‐regulated in male but not in female lungs.

Conclusions

Antenatal nicotine exposure results in fetal programming of abnormal lung development linking maternal nicotine use to increased susceptibility of interstitial pulmonary fibrosis in adult male offspring lung in a sex‐dependent manner.

Supported in part by NIH grant HL83966
Insulin Like Growth Factor‐1α Stimulates Differentiation of ATII Cells to ATI‐Like Cells Through Wnt5a
Authors: Ghosh Manik C , Gorantla Vijay , Makena Patrudu S , Luellen Charlean , Sinclair Scott E , Waters Christopher M
Published: June 2021

Alveolar type II (ATII) epithelial cells are important in the repair of the lung following acute lung injury. ATII cells have the capability to proliferate and differentiate into alveolar type I (ATI) cells in vivo, and can differentiate into an ATI‐like phenotype in vitro. The mechanisms responsible for transdifferentiation are not well understood. In this study we investigated the mechanisms by which insulin‐like growth factor 1‐α (IGF‐1α), which is increased in bronchoalveolar lavage fluid in patients with acute respiratory distress syndrome, promotes rat ATII to ATI differentiation in primary cultures. Gene expression of IGF‐1α was significantly increased in ATI‐like cells compared with ATII cells. Treatment of ATII cells with recombinant IGF‐1α accelerated the transition of ATII to ATI‐like cells, and this effect was inhibited by the IGF‐1α receptor blocker P40. Knockdown of Wnt5a using siRNA slowed the differentiation, as indicated by changes in the expression of ATII and ATI markers. These results suggest that IGF‐1α promotes differentiation of ATII to ATI cells through the non‐canonical Wnt pathway.
Proliferation of human alveolar epithelial cells down‐regulates ACE‐2 activity
Authors: Dang Vinh Xuan , Uhal Bruce David
Published: June 2021

In pulmonary fibrosis, a profibrotic effect is mediated by angiotensin II (ANGII). The accumulation of ANGII is limited by its conversion to angiotensin 1‐7 (ANG1‐7) by angiotensin converting enzyme‐2 (ACE‐2). Previous work from this lab demonstrated that ACE‐2 is protective against fibrogenesis, but down‐regulated in human and experimental lung fibrosis. ACE‐2 is expressed primarily by alveolar epithelial cells (AECs), which are quiescent in normal lung but proliferate in lung fibrosis. On this basis, we hypothesized that ACE‐2 activity would be dependent on cell cycle progression. It was predicted that ACE‐2 activity would be greater in quiescent cultures of human AECs (A‐549) compared to their proliferating counterparts. ACE‐2 activity was measured by assay with an ACE‐2 fluorogenic substrate and normalized to protein concentration. A competitive inhibitor for ACE‐2, Dx600, was co‐administered to analyze the specificity. In A‐549s, ACE‐2 activity was five‐fold greater in quiescent cells than proliferating cells (p = 0.0008). Inhibition of ACE‐2 with Dx600 demonstrated high specificity of the fluorogenic substrate. A similar effect was also seen in mouse lung epithelial cells (MLE‐12) with similar statistical significance (p < 0.001). These data show that proliferation of AECs down‐regulates ACE‐2 activity as a normal but unfortunate consequence of AEC proliferation during fibrogenesis. This cell culture model can be used to investigate the molecular control of the down‐regulation.

Support: PHS HL‐45136
TRPC1 and Orai1 interact with STIM1 and Mediate Capacitative Calcium Entry Activated by Acute Hypoxia in Mouse Pulmonary Arterial Smooth Muscle Cells
Authors: Ng Lih Chyuan , O'Neill Kathryn G. , French Dominique , Airey Judith A. , Singer Cherie A. , Tian Honglin , Shen Xiao-Ming , Hume Joseph R.
Published: June 2021

The present study aimed to determine if TRPC1 and Orai1 interact with STIM1 and mediate CCE caused by acute hypoxia in mouse PASMCs. In primary cultured PASMCs loaded with fura‐2, acute hypoxia caused a transient followed by a sustained rise in intracellular Ca2+ concentration ([Ca2+]i). Acute hypoxia also increased the rate of Mn2+ quench of fura‐2 fluorescence that was inhibited by SKF 96365, Ni2+, La3+ and Gd3, exhibiting pharmacological properties characteristic of CCE. The nifedipine‐insensitive rise in [Ca2+]i and the increase in Mn2+ quench rate were both inhibited in cells treated with TRPC1 antibody, in cells transfected with STIM1 siRNA and cells transfected with Orai1 siRNA. Moreover, overexpression of STIM1 resulted in a marked increase in [Ca2+]i and Mn2+ quench rate caused by acute hypoxia, and they were reduced in cells treated with TRPC1 antibody and cells treated with Orai1 siRNA. Furthermore, TRPC1 and Orai1 co‐immunoprecipitate with STIM1 and the precipitation levels of TRPC1 and Orai1 were increased in cells exposed to acute hypoxia. Immunostaining showed co‐localizations of TRPC1‐STIM1 and Orai1‐STIM1, and the co‐localizations of these proteins were more apparent in acute hypoxia. These data provide direct evidence that TRPC1 and Orai1 channels mediate CCE through activation of STIM1 in mouse PASMCs in acute hypoxia. [Supported by HL49254 (JR Hume) and AHA Scientist Development Grant (LC Ng)]
Apyrase (APY) inhibits mechanical activation of nodose C‐fibers in guinea pig lung
Authors: Weigand Letitia A. , Undem Bradley J.
Published: June 2021

Vagal sensory C‐fiber activation in the lungs leads to symptoms of airway diseases. The two subtypes of vagal respiratory C‐fibers have cell bodies in either the nodose or jugular ganglia. We have shown that nodose, but not jugular C‐fibers, are strongly activated by histamine (HA) and methacholine (MCh), responses that are inhibited by blocking smooth muscle contraction (ML‐7 or isoproterenol), and nearly abolished by two structurally unrelated P2X3‐P2X2/3 receptor antagonists (AF‐353 or TNP‐ATP). We hypothesized that the C‐fibers are activated by ATP released during airway smooth muscle contraction, and independently addressed this hypothesis using the enzyme APY to metabolize ATP. We recorded action potential (AP) discharge from single nodose C‐fibers in an ex vivo isolated perfused guinea pig lung nerve preparation exposed to HA (30 μM) before and after APY. APY alone (5 or 10 U/ml) had no effect on HA‐induced AP discharge (n=4 P>0.1). We hypothesized that ATP hydrolysis produced adenosine (ADO; activates nodose C‐fibers). Blocking ADO receptors (A1, DPCPX 100 nM; A2, SCH58261 100 nM) had no effect on HA‐induced AP discharge (n=3, P>0.1), but in the presence of ADO receptor blockade, APY (5 U/mL) nearly abolished the response to HA (14 ± 3 to 3 ± 0 Hz, P <0.05 n=4). These results provide additional evidence that ATP released from the tissues plays a pivotal role in the mechanical activation of nodose C‐fibers.
Angiotensin type 1A receptors (AT1a) and reactive oxygen species (ROS) mediate nuclear factor‐κ‐B (NF‐κB) activation in the subfornical organ (SFO) during slow‐pressor Angiotensin‐II (Ang‐II) hypertension
Authors: Li Anfei , Young Colin N. , Davisson Robin L.
Published: June 2021

We recently showed that NF‐κB is activated in the SFO of the brain during slow‐pressor Ang‐II hypertension (HTN). The molecular mechanisms contributing to NF‐κB activation, however, remain unclear. We tested the hypothesis that NF‐κB activation in the SFO during slow‐pressor Ang‐II HTN is mediated by AT1a receptors and ROS. Male AT1a flox mice underwent SFO‐targeted injections of an adenovirus encoding firefly luciferase downstream of NF‐κB response elements, in conjunction with adenoviral delivery of Cre‐recombinase (Cre) for selective deletion of AT1a or control LacZ. After recovery, osmominipumps were implanted for slow‐pressor Ang‐II infusion (600ng/kg/min), and NF‐κB activity in the SFO was measured daily using in vivo bioluminescence imaging. A surge in SFO‐NF‐κB activity was seen at day 5 of slow‐pressor Ang‐II infusion and this was significantly reduced by deletion of AT1a (LacZ 1.8 ± 0.7 vs Cre −0.2 ± 0.1 photons/s fold baseline, p<0.05, n=6). In a separate cohort of mice, scavenging of superoxide in the SFO via adenoviral overexpression of CuZnSOD also prevented the prehypertensive surge in NF‐κB activity (Day 5: LacZ 1.5 ± 0.7 vs CuZnSOD −0.5 ± 0.1 photons/s fold baseline, p<0.05, n=9–10). These findings suggest that AT1a redox‐sensitive mechanisms are key steps mediating SFO‐NF‐κB activation during slow‐pressor Ang‐II

HTN. HL864624
Anoxia‐induced respiratory arrest and autoresuscitation in rat pups exposed to dietary tryptophan deficiency
Authors: Corcoran Andrea E , Kinney Hannah C , Nattie Eugene E
Published: June 2021

Many Sudden Infant Death Syndrome cases are associated with abnormalities in the medullary serotonergic system. We hypothesize that these abnormalities impair infants from adequately responding to stresses, such as hypoxia and hypercapnia (asphyxia), which may be a consequence of rebreathing exhaled gases when in the prone positition. Asphyxia initiates a series of events that lead to a reflex known as autoresuscitation. To examine the role of a moderate depletion of serotonin (5HT) on autoresuscitation, we studied pups born from Sprague‐Dawley dams fed a diet partially deficient in tryptophan (~45%). Using head‐out plethysmography, we identified the cardiorespiratory responses to repeated episodes of anoxia (97% N2:3% CO2) and the ability to autoresuscitate from anoxia‐induced respiratory arrest. At each age studied (P5, P8, P12) tryptophan deficient pups had a similar level of autoresuscitation failure as control. We then examined the effect of “pretreatment” with prior post‐natal chronic intermittent hypoxia (CIH) on the ability to autoresuscitate. CIH increased the ability of control pups to autoresuscitate but had no effect on survival in tryptophan deficient pups (P=0.042; Chi‐square). This suggests that CIH improves the ability to autoresuscitate from anoxia‐induced respiratory arrest, likely through a 5HT dependent mechanism. (NIH National Institute of Child Health and Development HD36379)
Comparative effects of fatty acid synthase inhibitors, C75 and cerulenin, on brown adipose tissue thermogenesis in Wistar rats
Authors: Cassolla Priscila , Garófalo Maria Antonieta R. , Kettelhut Isis C. , Navegantes Luiz C. C.
Published: June 2021

Fatty acid synthase (FAS) inhibitors, C75 and cerulenin, are anorexigenic compounds that promote weight loss. In previous studies, we demonstrated that the central injection of C75 increased the sympathetic outflow to brown adipose tissue (BAT) suggesting activation of the non‐shivering thermogenesis. In the present study, we asked whether or not cerulenin, a natural FAS inhibitor, would have similar effects than C75 on BAT. Wistar rats (~230 g) were submitted to BAT sympathetic denervation following by stereotaxic surgery and implant of i.p. or BAT temperature transponders. Seven days later, C75 (150μg/7.5μL), cerulenin (150μg/7.5μL) or RPMI was administered intracerebroventricularly in 24h‐fasted animals. Both C75 and cerulenin increased core body temperature but did not affect the locomotor activity. While the thermal effect of C75 lasted for 9 h (start at 30 minutes of injection), the effect of cerulenin lasted only for 2h (start at 4 h of injection). C75 and cerulenin also increased BAT temperature for 7 h (start at 1 h of injection) and 2 h (after 3 h of injection), respectively. The thermal effects of both FAS inhibitors were abolished by BAT sympathetic denervation. In contrast to C75, cerulenin did not change food intake. In conclusion, the central effects of C75 were faster and stronger than cerulenin on SNS‐activated BAT thermogenesis. Financial support: FAPESP (08/06694‐6)
CARDIOVASCULAR EFFECTS AFTER GLUTAMATE MICROINJECTION INTO THE RVLM OF MSG OBESE CONSCIOUS RATS
Authors: Martins-Pinge Marli Cardoso , Cunha Natália Veronez
Published: June 2021

Obesity is a precursor condition of several diseases, among them hypertension. The sympathoexcitatory neurons of the rostral ventrolateral medulla (RVLM), which directly innervate sympathetic preganglionic neurons in the intermediolateral cell columns of the spinal cord, are a possible target for cardiovascular modulation by obesity, and glutamatergic neurotransmission has an important role. Thus, the aim of this study was to determine the effect of glutamate microinjection into the RVLM in control and MSG obese rats. Obesity was induced by intradermal administration of 4 mg/g of monosodium glutamate (MSG) or hyperosmotic saline (control = CTR) in the first 5 days of life in male Wistar rats. At 90 days old, the animals were submitted to guide cannulae implantation to the RVLM for unilateral microinjection of L‐glutamate (5 nmol/100 nl) in conscious state for recording for mean arterial pressure (MAP) and heart rate (HR). There was no difference in MAP and HR at baseline among the experimental groups (CTR: 112 ± 2 mmHg, 362 ± 14 bpm; MSG: 106 ± 3 mmHg, 389 ± 13 bpm). Unilateral microinjection of l‐glutamate produced an increase in MAP in CTR and MSG groups, however, the magnitude of this response was higher in obese rats (CTR: 44 ± 3; MSG: 56 ± 2, p=0,02). The excitation of the RVLM with glutamate decreased HR to levels that were not statistically different between groups (CTR: −26 ± 19 bpm; MSG: −45 ± 37 bpm). The present results suggest that MSG obese rats may have elevated sympathetic nerve activity originated from the activity of RVLM neurons.