bluuebunny/crossref_metadata_2025 huggingface dataset:
Photo‐crosslinking Reveals Unique Features of the Sigma‐1 Receptor Ligand Binding Region(s)
Authors: Chu Uyen B. , Ramachandran Subramaniam , Hajipour Abdol R. , Ruoho Arnold E.
Published: June 2021

We have recently found that endogenous sphingolipids including sphingosine and sphinganine interact with the sigma‐1 receptor. To study the interaction of these lipid molecules with the receptor, we used a series N‐alkylamines and their phenylpropyl‐ as well as nitrophenylpropyl‐ derivatives as surrogates for sphingosine. Surprisingly, N‐(3‐(4‐nitrophenyl)propyl)‐N‐alkylamines, revealed a unique property of the sigma‐1 receptor whereby upon UV irradiation of the receptor in the presence of excess compounds, two sigma‐1 receptor populations were generated. The first population migrated as a 26 kDa and the second as 23 kDa on an SDS polyacrylamide gel with a ratio of 1:1. Using anti‐sigma‐1 receptor antibody, we also detected this phenomenon in both guinea pig liver membranes and COS‐7 cells overexpressing the sigma‐1 receptor. Generation of these two sigma‐1 receptor populations was dependent on photolysis of N‐alkylamine derivatives of 12 carbons or longer and was protectable by haloperidol. The 23 kDa population was not formed as a result of photo‐chemical cleavage of the sigma‐1 receptor since both of the N‐ and C‐termini are presence as confirmed by N‐terminal sequencing and western analysis using an anti‐C‐term hexahistidine antibody. Sequence specific antibodies against amino acids 143 – 169 of the sigma‐1 receptor showed that in the 23 kDa, the N‐alkylamine photoprobe blocked the antibody epitope of this region. Additionallly, MALDI‐TOF results showed that there is a 318 Da shift of the peptide corresponding to amino acids 143 – 175, suggesting a covalent cross‐linking of photoprobe to the pure protein. Together, these data reveal unique features of the sigma‐1 receptor binding region(s).
Role of Growth factor receptor transactivation in high glucose‐induced increased levels of Gq/11α and signaling in vascular smooth muscle cells
Authors: Descorbeth Magda , Anand‐Srivastava Madhu B.
Published: June 2021

We have recently shown that high glucose (HG) increased the expression of Gq/11α, PLCβ and mediated signaling in A10 vascular smooth muscle cells (VSMC). Since HG has been shown to increase growth factor receptor (GFR) activation, we investigated the role of epidermal growth factor receptor (EGF‐R) and platelet‐derived growth factor receptor (PDGF‐R) transactivation in HG‐induced enhanced expression of Gq/11α and PLCβ. Pre‐treatment of A10 VSMC with HG (26 mM) for 3 days, increased the levels of Gqα, G11α, PLCβ‐1 and PLCβ‐2 proteins as determined by Western blotting which were restored to control levels by AG1478, an inhibitor of EGF‐R, AG1295, an inhibitor of PDGF‐R and PP2, an inhibitor of c‐Src but not by PP3. In addition, endothelin‐1 (ET‐1)‐stimulated production of IP3 that was enhanced by HG was also restored towards control levels by AG1478, AG1295 and PP2. HG also increased the phosphorylation of EGF‐R and PDGF‐R which was abolished by AG1478, AG1295 and PP2. Furthermore, HG‐induced enhanced levels of Gqα, G11α and PLCβ were also attenuated by inhibition of MAP Kinase, PI3‐Kinase and c‐Src. In addition, GFR inhibitors also attenuated HG‐induced enhanced phosphorylation of ERK1/2 and AKT. These results suggest that c‐Src‐induced transactivation of growth factor receptor contributes to the high glucose‐induced enhanced expression of Gq/11α/PLC and ‐mediated cell signaling through MAPK/PI3K pathway.

This work was supported by Canadian Institutes of Health Research grant
Attenuation of β‐cell death by P2Y6 receptor agonist: A novel drug target for diabetes
Authors: Balasubramanian Ramachandran , de Castro Sonia , Maruoka Hirsohi , Jacobson Kenneth A
Published: June 2021

Apoptosis plays a key role in the reduction of β‐cell mass and has been proposed to mediate β‐cell reduction in type 1 and type 2 diabetes. Hence drugs which decrease blood glucose as well as protect β‐cells are required. In this context, we are evaluating the insulin secreting efficacy and cytoprotective nature of P2Y6 receptor agonists in the present study. MIN6 cells grown to 80% confluency, were induced for insulin secretion in the presence of 16.7 mM glucose, with or without P2Y6 agonists such as UDP, Up3U, and other novel P2Y6 agonists. MIN6 cells were treated with 50 ng/ml TNF‐α for 24 h to induce apoptosis in the presence of P2Y6 agonists. The efficacy of P2Y6 agonist to attenuate apoptosis was determined by cell cycle analysis using flow cytometry. DNA fragmentation was detected by TUNEL assay based on 5‐bromo‐2’‐deoxyuridine(BrdU) labeling. Insulin secretion in MIN6 cells was significantly potentiated by P2Y6 agonists. Also P2Y6 agonists significantly mitigated TNF‐α induced cell death. These P2Y6 agonists that are both insulin secretagogues and cytoprotective could be developed as a therapeutic intervention for diabetes mellitus. This work was supported by the Intramural Research Program of NIDDK, National Institutes of Health, MD, USA.
Contamination of the Norepinephrine Pro‐Drug Droxidopa by Dihydroxyphenylacetaldehyde
Authors: Holmes Courtney S , Goldstein David S
Published: June 2021

Background

Droxidopa (3,4‐threo‐L‐dihydroxyphenylserine, L‐DOPS) is a norepinephrine (NE) pro‐drug under development to treat orthostatic hypotension. 3,4‐Dihydroxyphenylacetaldehyde (DOPAL) is a toxic catecholaldehyde.

Methods

Thirteen subjects took 400 mg Droxidopa orally, with blood sampled at baseline and 1, 2, 3, 6, 24, and 48 hours after drug administration and assayed for plasma L‐DOPS, NE, and DOPAL. Droxidopa in acidic solution was vacuum centrifuged for 1 hour at 30 degrees centigrade.

Results

Droxidopa contained 0.01% DOPAL. At 6 hours, all subjects had DOPAL detected in plasma (1.89 nmol/L, p=0.0001). Across the sampling times plasma DOPAL correlated with plasma L‐DOPS (r=1.00). The mean increment in plasma DOPAL averaged more than 4 times the mean increment in plasma NE (0.39 nmol/L). In 2 patients with Parkinson disease and orthostatic hypotension, DOPAL was detected at baseline (0.12 nmol/L) and increased by about 70‐fold after Droxidopa. After 1 hour of vacuum centrifugation, L‐DOPS was converted completely to DOPAL.

Interpretation

Droxidopa is contaminated with DOPAL. After Droxidopa ingestion DOPAL is detectable in plasma. Droxidopa is converted completely to DOPAL under conditions resembling those in the stomach.
Andrographolide as a template in the synthesis of new anticancer agents with potentially novel molecular targets
Authors: Stanslas Johnson , Lim Siang Hui , Wong Charng Choon , Sagineedu Sreenivasa Rao , Lajis Nordin Haji
Published: June 2021

Using andrographolide (AGP) as a template, a series of semisynthetic analogues was synthesised for the discovery of new anticancer agent(s) with improved antitumour selectivity and potency. SRJ09, a lead compound displayed remarkable activity in the National Cancer Institute (NCI) of USA in vitro anticancer screen, particularly against breast and colon cancers. The treatment of MCF‐7 breast and HCT‐116 colon cancer cells with SRJ09 induced a G1 phase cell cycle arrest through the down‐regulation of CDK‐4 expression with concomitant up‐regulation of p21 expression and without affecting the cyclin D1 expression. SRJ09 induced apoptotic cell death in both cells in a p53‐ and bcl‐2‐independent manner, implicating extrinsic apoptotic pathway. The compound showed impressive in vivo activity by delaying HCT‐116 tumour xenograft growth in nude mice. Recently, a derivative of SRJ09, SRS07 showed vast improvement in the NCI anticancer activity screen compared with the parent compound. Additionally, NCI's in silico COMPARE and SOM analyses revealed that the new AGP derivatives have potentially novel molecular target(s). These findings although preliminary they are highly suggestive SRS07 have a great potential to be developed into a clinical anticancer agent. This research is funded by the Ministry of Science, Technology and Innovation of Malaysia (MOSTI) under the IRPA and Science Fund grant schemes.
Studies on the Anti‐inflammatory, Antinociceptive and Antipyretics activities of extract from Leaves of Labisia pumila
Authors: Ikujuni Catherine Moyinoluwa , Okechukwu Patrick Nwabueze , Rajagopal Mariani , Panrengan Kumutamalar , Akowuah Gabriel Akyirem
Published: June 2021

The dichloromethane (DCM) extract of Labisia pumila leaves were investigated for its anti‐inflammatory (acetic acid‐induced writhing and carrageenan‐induced paw oedema test), antinociceptive (hot‐plate and formalin test) and anti‐pyretics (yeast‐induced hyperpyrexia test) effects. The phytochemical screening of the extract was also investigated. In the anti‐inflammatory test, DCM extract (100 mg/kg) inhibited significantly the acetic acid‐induced writhing by 78.34% in relation to positive control (aspirin 100 mg/kg) value 88.93% (p<0.05), while the carrageenan‐induced paw oedema was reduced by 50% in relation to positive control (indomethacin 10 mg/kg) value 38.24% (p<0.05) in the experimental rat model. In the antinociceptive test, the extract (100 mg/kg) significantly reduced the licking time in both the early and late phases of the formalin test by 68.41 and 49.17%, in relation to positive control (morphine 5 mg/kg) value 75.17 and 100% (p<0.01), respectively. The extract (100mg/kg) also showed significant effects on hot plate test by 48.75% (p<0.01). Treatment with DCM extract (100 mg/kg) showed a significant reduction in yeast‐induced pyrexia in the fourth hour by 5.4% in relation to positive control (aspirin 100 mg/kg) value 4.3% (p<0.01). Phytochemical screening of the extract indicated the presence of flavonoids, steroids and saponins. The present study confirmed that extracts from the leaves of L. pumila exhibited anti‐inflammatory, antinociceptive and anti‐pyretic activities and these effects might be due to the observed phytochemicals.
VEGF‐A165b is cytoprotective for ARPE‐19 cells indicating a potential role in AMD therapy
Authors: Beazley‐Long Nicholas , Churchill Amanda J , Harper Steven J , Bates Dave O
Published: June 2021

Age‐related Macular Degeneration (AMD), the most common cause of blindness in the developed world is treated by intravitreal drugs that target Vascular Endothelial Growth Factor (VEGF). The aberrant angiogenesis of neovascular AMD through the Bruch's membrane and into the subretinal space disrupts the retinal pigmented epithelial (RPE) layer and photoreceptors. VEGF‐A165 is cytoprotective for RPE cells and systemic toxicities to high‐dose anti‐VEGF therapy are emerging. VEGF‐A165b, arising from alternative splicing of exon 8 of the vegf gene is anti‐angiogenic and has recently been ascribed cytoprotective qualities. To determine whether VEGF‐A165b was cytoprotective for the human RPE cell line ARPE‐19, hydrogen peroxide (H2O2) and 7‐ketocholesterol induced cytotoxicity, thought to contribute to the progressive degeneration of the RPE layer in AMD, was measured using a cell viability (MTT) and cytotoxicity assay (LDH). 2.5 nM VEGF‐A165b reduced cytotoxcity for ARPE‐19 cells to H2O2 (400 μM; 24 h) by 49±16%, to 7‐ketocholesterol (16–20 μM; 24 h) by 85±2% and increased viability by 138±16%. The anti‐angiogenic action and RPE cytoprotection support VEGF‐A165b as a promising candidate for future AMD therapy. Research funded by the Wellcome Trust.
Endothelin‐A Receptor Blockade Improves Angiogenic Signaling in the Stenotic Kidney
Authors: Chade Alejandro R , Kelsen Silvia , Bailey James E , Fails Fredrick
Published: June 2021

Background

Chronic renal artery stenosis (RAS) induces endothelin (ET)‐1 up‐regulation and via the ET‐A receptor can promote renal injury. Chronic RAS promotes renal microvascular (MV) damage and loss but the role of the ET/ET‐A is unknown. Thus, we hypothesized that ET‐A blockade in RAS will preserve the MV architecture by improving the angiogenic signaling in the stenotic kidney.

Methods

RAS was induced in 12 pigs and observed for 6 weeks. In 6 of them, ET‐A blocker was chronically administered (RAS+ET‐A, 0.75 mg/kg/day). Additional animals were used as normal controls (n=6). Single‐kidney MV architecture and function was quantified using CT and renal angiogenic and apoptotic signaling determined by western blot.

Results

ET‐A blockade in RAS improved the renal expression of VEGF and augmented downstream mediators such as angiopoietin‐1 and 2, its receptor Tie‐2, and eNOS. Interestingly, it also decreased Bax/Bcl‐2 ratio, suggesting attenuated apoptotic signaling in the stenotic kidney. Consequently, MV density and function in the RAS+ET‐A kidney was improved.

Conclusion

Chronic ET‐A blockade improved angiogenic and decreased apoptotic signaling in the stenotic kidney. These ultimately preserved the renal MV architecture and function in the stenotic kidney, supporting a novel renoprotective role of ET‐A blockade in chronic RAS.

Support: AHA‐SDG‐0830100N
Perfusion Restoration Following Femoral Artery Occlusion is Markedly Inhibited in Mice with MMP9 Deficient Bone‐Marrow Derived Cells
Authors: Meisner Joshua K , Bajikar Sameer S , Price Richard J
Published: June 2021

Numerous studies have shown bone marrow‐derived cells (BMCs) play a critical role in orchestrating arteriogenesis. Bakker et al (2008) recently showed that BMCs, monocytes in particular, orchestrate arteriogenesis by allowing for outward or inward remodeling depending on arterial tone. We propose that one mechanism through which BMCs perform this accommodating function is through the secretion of matrix metalloproteases, resulting in a number of actions such as the breakdown of extracellular matrix (ECM) components. In particular, MMP9 is known to play an important role in vascular remodeling. Therefore, we hypothesized that the knockout of MMP9 specifically in BMCs will decrease arteriogenesis in a mouse model of hindlimb ischemia. To test our hypothesis, we created bone marrow (BM) chimeric mice by lethally irradiating host wild‐type (WT) mice and then reconstituting them with BM from either WT (WT→WT, control) or MMP9−/− (MMP9→WT) mice. Femoral artery ligation was then performed to stimulate arteriogenesis in the vessels of the upper hindlimb. Leg revascularization was monitored non‐invasively using laser Doppler perfusion imaging over the course of 14 days. MMP9→WT mice show a significant decrease in revascularization versus control mice at all time points after day 2. These and additional preliminary findings in collateral vessel cross‐sections and whole mounts demonstrate that BMC‐specific MMP9 expression is critical for normal perfusion restoration through arteriogenesis. Supported by NIH R01‐HL074082‐04.
Meningeal vascular response to estrogen‐based therapy in post ovariectomized pigs
Authors: Glinskii Olga , Huxley Virginia , Rubin Leona , Abraha Tsghe , Glinsky Vladislav
Published: June 2021

Loss of gonadal function in women is associated with increased incidence of stroke, cranial aneurysms, and neurodegenerative disease. Those sequelae infer that intracranial vascular adaptation at the onset of reproductive senescence confers enhanced cerebrovascular risk. Our data from in vivo pig model show that following ovariectomy (OVX), meningeal vasculature undergoes substantial remodeling characterized by loss of capillaries, increase in average microvessel size and augmented permeability associated with estrogen receptor (ER) alpha dependent decrease in angiopoietin‐1 expression. Even 2 months post OVX, vascular networks remain destabilized and prone to further remodeling. Mechanistically, this process is regulated in part by ER‐related pathways and, therefore, is estrogen‐dependent. However, effects of estrogen (E2) based therapy on such remodeling depend strikingly on the treatment regimen. We show that flat‐dose E2 therapy fails to prevent vascular remodeling, while E2 regimens mimicking natural estrous cycle‐associated E2 fluctuations prevent post OVX microvascular alterations. It appears that, as opposed to conventional flat‐dose regimens, pulsed E2‐based therapy could present a compelling strategy for preventing massive intracranial vascular remodeling during reproductive senescence compromising functional and structural integrity of meningeal microvasculature.

Supported by AHA National SDG 0830287N, NIHHL078816, NASA NNJ05HF37G, NIHC06RR017353
Aging‐induced changes in angiotensin II‐induced contractions and tachyphylaxis of isolated carotid arteries
Authors: Koller Akos , Toth Peter , Rozsa Bernadett , Cseplo Peter , Hamar Janos , Vamos Zoltan
Published: June 2021

Angiotensin II (Ang II) is a potent vasoconstrictor, yet the potential aging‐ induced changes in the magnitude of responses have not yet been explored. We hypothesized that aging induces changes in the magnitude of vasomotor responses to Ang II. Thus carotid arteries from newborn (8 days: 8d), young (2 months: 2m) and old (24 months: 24m) rats were isolated and placed in a wire myograph to measure changes in their isometric tension. Two dose response curves to Ang II were obtained in a sequential manner (1‐, and 2‐administration (Adm). Contractions of vessels to 1‐Adm of Ang II in 8d vessels were significantly less compared to those of 2m and 24m, which were similar in magnitude (1.40±0.2 mN and 5.2±0.6 and 5.7±0.39 mN, respectively). Compared to the 1‐Adm, contractions of vessels to 2‐Adm of Ang II were significantly reduced in 2m and 24m vessels (4.4±0.54 and 0.5±0.19mN), whereas it did not change in vessels of 8d rats (1.28±0,2 mN).

These findings suggest that in carotid arteries 1) Ang II‐induced contractions increase with age, 2) repeated administration of Ang II elicits tachyphylaxis and the magnitude of tachyphylaxis increases with aging.

(Hungarian Nat. Sci. Res. Fund.‐OTKA K71591, K67984, AHA Founders Aff., 0555897T)
Resveratrol induces mitochondrial biogenesis in endothelial cells
Authors: Csiszar Anna , Zhang Hanrui , Zhang Cuihua , Ungvari Zoltan
Published: June 2021

Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mtDNA content, up‐regulated protein expression of electron transport chain constituents and induced mitochondrial biogenesis factors (PGC‐1á, Nrf‐1, Tfam). SIRT1 was induced and eNOS was up‐regulated in a SIRT1‐dependent manner. Knockdown of SIRT1 (siRNA) or inhibition of NO synthesis prevented resveratrol‐induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. We propose that SIRT1, via a pathway that involves up‐regulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.
Rac2 in murine pulmonary microvascular endothelial cells (ECs)
Authors: Gomez John Clifford , Doerschuk Claire M.
Published: June 2021

Rac2 is a small GTPase in the Rho family. The lungs of uninjured Rac2−/− mice show enhanced plasma flux. This study determined if Rac2 is expressed and regulates phosphorylation of cytoskeletal proteins in ECs. Real time PCR showed that ECs isolated from murine lung digests by negative selection of CD45+ cells (leukocytes) and positive selection of CD105+ ECs express Rac2 mRNA. An 11 amino acid region in the C‐terminus of murine Rac2 was used to generate a rabbit polyclonal anti‐Rac2 antibody (Pacific Immunology). This antibody recognized Rac2 (21kDa) on immunoblots of murine Rac2+/+ but not −/− ECs and splenocytes, and not human Rac2 in HL60 cells. Protein arrays assessing 95 proteins associated with the cytoskeleton (cytoskeleton phospho antibody microarray, Full Moon Biosystems) showed that Rac2−/− ECs had increased expression of 2 proteins (caveolin‐1: 2.5 fold, LIMK1: 2.0 fold) and decreased expression or phosphorylation of 4 proteins (cortactin: 0.41, MEK1(Phospho‐Ser217): 0.06, MEK1(Phospho‐Thr291): 0.54, Src: 0.53, Vav(Phospho‐Tyr174): 0.54 fold). Thus, Rac2 mRNA and protein are expressed by ECs. Rac2 appears to regulate the cytoskeletal structure and function of ECs. Funded by HL 077370.
The essential role of inducible nitric oxide synthase for mobilization of bone marrow stem cells to the cochlea
Authors: Shi Xiaorui , Nuttall Alfred L.
Published: June 2021

Inducible nitric oxide synthase (iNOS) produces an essential signal for homing of circulating bone marrow derived stem cells (BMDSC) to target tissues. We find that the iNOS responsible for homing of BMDSC in the vascular “lateral wall” of the cochlea is from resident macrophages. The de novo macrophages appear to be essential for vascular repair in a model of inner ear ischemia produced by loud sound. The data show that mice deficient in iNOS (Nos2−/−) or mice treated with an iNOS‐inhibitor had a reduction of the mobilization of bone marrow progenitor cells to the cochlea and less hearing function recovery after acoustic trauma. Mechanistically, stromal cell‐derived factor‐1(SDF‐1) and its chemokine (CXC motif) receptor 4 (CXCR4) is required for stem cell mobilization, and was found to be reduced in the bone marrow of Nos2−/− mice and in animals treated with iNOS inhibitor. Loud sound induces iNOS expression in local perivascular cells (macrophages) which then becomes a major cell for NO production in the local microenvironment. These findings indicate that iNOS expressed by local resident macrophages influences the recruitment of BMDSC. The homing is abrogated by inhibition of SDF‐1 a/CXCR4 productions. Movement of BMDSC into the vascular area could be a basis of vascular remodeling and repair following loud sound damage.

Support: NIDCD DC 00888, NIDCD DC 00105, NIDCD DC 005983 (p30)
In vivo inhibition of Nuclear Factor of Activated T‐cells (NFAT) results in enhanced levels of anti‐inflammatory IL‐10 in the retina of diabetic mice
Authors: Zetterqvist Anna V , Nilsson‐Öhman Jenny , Kotova Olga , Nilsson‐Berglund Lisa M , de Frutos Garcia Sergio , McGuire Paul , Gonzalez‐Bosc Laura V , Gomez Maria F
Published: June 2021

Hyperglycemia is an important risk factor for the development of diabetic retinopathy. Recently we showed that high glucose activates NFAT in cerebral artery smooth muscle through local release of extracellular nucleotides, acting on P2Y receptors, leading to increased [Ca2+]i and calcineurin activation. NFAT activation promotes cell proliferation and the expression of inflammatory markers, such as IL‐6, COX‐2 and osteopontin, which are elevated in diabetic retinopathy. Here we investigate whether: 1) NFAT is activated by hyperglycemia in the wall of retinal microvessels (RM) and 2) inhibition of NFAT signaling may have a protective effect on the retina of diabetic mice. Using confocal immunofluorescence microscopy we show that NFATc3 is expressed in the endothelium of RM and readily activated by high glucose ex vivo and in vivo (intra peritoneal glucose tolerance test). This activation is inhibited by the NFAT blocker A‐285222 and by the ecto‐nucleotidase apyrase. Chronic hyperglycemia in STZ‐treated NFAT‐luc mice leads to increased NFAT transcriptional activity in isolated RM. Interestingly, two weeks after the first STZ injection, levels of the potent anti‐inflammatory cytokine IL‐10 were decreased in retina and this was prevented by treatment with A‐285222. Results suggest that NFAT inhibition may protect the retina of diabetic mice.

Funding: Swedish Heart & Lung Foundations, RAC HSC‐UNM 624195
Serine 225 phosphorylation governs the localization and function of sphingosine kinase 1 in resistance arteries
Authors: Lidington Darcy , Peter Bernhard Friedrich , Meissner Anja , Kroetsch Jeff , Pitson Stuart , Pohl Ulrich , Bolz Steffen‐Sebastian
Published: June 2021

Sphingosine kinase 1 (Sk1), which synthesizes the vasoconstrictor sphingosine‐1‐phosphate (S1P), must translocate from the cytosol to the plasma membrane in order to access its substrate. We aimed to characterize the serine 225 phosphorylation motif as the molecular switch that regulates the pressure‐dependent activation/translocation of Sk1 in hamster gracilis muscle resistance arteries. Pressure elevation stimulated the phosphorylation of ERK1/2, an upstream activator of Sk1, and the translocation of Sk1 to the plasma membrane. Functionally, preventing Sk1 activation, either by the expression of a non‐phosphorylatable mutant (Sk1S225A) or by ERK1/2 inhibition, reduced resting and myogenic tone, resting Ca2+, pressure‐induced Ca2+ elevation and Ca2+ sensitivity. The effects of Sk1S225A expression on Ca2+ were restored by forcing its localization to the plasma membrane, but all other parameters remained inhibited. Forcing wild‐type Sk1 to the plasma membrane inhibited the same parameters. These discrete effects are consistent with cell surface receptor‐mediated effects and intracellular effects of S1P. Accordingly, S1P2 receptor inhibition attenuated myogenic tone without effect on Ca2+. We conclude that: (i) translocation and precise subcellular positioning of Sk1 is essential for full Sk1 function; and (ii) two distinct S1P pools contribute to the maintenance of vascular tone.
Mechanical Force Induced Calcium Signals are Extracellular Matrix (ECM) Specific in Arteriolar Vascular Smooth Muscle Cells (VSMC)
Authors: Sun Zhe , Li Zhaohui , Meininger Gerald A.
Published: June 2021

It has been proposed that integrin‐mediated mechanisms contribute importantly to mechano‐regulation of Ca2+in VSMC. An integrated atomic force (AFM)‐confocal microscopy system was used to investigate the relationship between pulling force applied through integrin‐ECM protein adhesions and Ca2+ signals in VSMCs isolated from rat cremaster feed arterioles. AFM attached microbeads (5 μm) were coated with fibronectin (FN), collagen type I (CNI) or type IV (CNIV) and positioned on a VSMC for 20 min resulting in formation of a focal adhesion between the cell and microbead. Application of step increases in pulling force (800~3000 pN) to FN or CNI adhesions induced Ca2+transients that increased in frequency and intensity with applied force. An increase in the total area of the cell showing transients was only observed with FN at high force (3000 pN). No changes in Ca2+ behavior were observed during pulling through CNIV or albumin coated beads. However, detachment of the CNIV bead from cell surface increased the area of Ca2+ transient activity, which was not observed with FN‐ and CNI‐coated beads. These data suggests that mechanotransmission through FN‐, CNI‐ and CNIV‐ alters Ca2+ signaling in VSMC in a force and ECM dependent manner. (Supp. by NIH HL58690 to G.A. Meininger and AHA 0765481Z & 0835676N to Z. Sun)
Chronic CNS actions of adiponectin on appetite, metabolism and blood pressure
Authors: Bassi Mirian , Do Carmo Jussara M , Silva Alexandre A , Hall John E
Published: June 2021

Acute studies suggest that adiponectin may reduce sympathetic activity and blood pressure (BP) via its action on the central nervous system (CNS). We tested if chronic intracerebroventricular (ICV) infusion of adiponectin at 2 doses (1 or 7 μg/day) in Sprague‐Dawley rats fed high fat diet (HFD) for 8 weeks, and at the high dose (7 μg/day) in spontaneously hypertensive rats (SHR), a model of hypertension associated with sympathetic overactivity, evoked chronic reductions in BP and heart rate (HR). We also determined if chronic ICV adiponectin infusions alter appetite, whole body oxygen consumption (VO2), and insulin and leptin levels. Neither dose of adiponectin infused ICV for 7 days altered BP or HR in the HFD group (115±2 to 112±2 mmHg and 384±6 to 379±6 bpm at 1 μg/day; 109±3 to 111±3 mmHg and 366±5 and 367±5 bpm at 7 μg/day). The higher dose slightly reduced food intake (14±1 to 11±1 g/day), whereas VO2, insulin and leptin levels were not affected by the treatment. In SHRs, ICV adiponectin infusion reduced appetite (22±2 to 12±2 g/day) and insulin levels (~55%), but did not alter BP (162±4 to 164±3 mmHg) or HR (312±5 to 322±8 bpm). These results suggest that adiponectin, acting via its direct actions on the CNS, has a small effect to reduce appetite and insulin levels. However, our results indicate that, at the doses used in this study, adiponectin has no long‐term action to reduce BP or HR, or to alter whole body metabolic rate.
Cardiovagal baroreflex sensitivity is not correlated to sympathetic baroreceptor sensitivity within healthy, young individuals
Authors: Dutoit Andrea , Hart Emma , Charkoudian Nisha , Wallin BG , Curry Tim , Joyner Michael
Published: June 2021

The purpose of this study was to evaluate the relationship between the cardiac and sympathetic baroreflex sensitivities within healthy, young individuals. The gains in cardiac (as measured by changes in R‐R interval, RRI, and heart rate, HR) and sympathetic (as measured by changes in muscle sympathetic nerve activity, MSNA) baroreflex during rapid changes in arterial blood pressure were compared in 27 normotensive individuals (18 men and 9 women; age 24 yrs ±1.24; body mass index 24 ±0.51 cm/kg2, mean ±SEM). Heart rate, arterial blood pressure (brachial artery catheter), and peroneal MSNA were recorded under resting conditions (HR 58 ±1.6 bpm; SBP 124 ±2.7 mmHg; DBP 71 ±1.7 mmHg; MAP 87 ±1.7 mmHg; MSNA BF 17 ±1.6 bursts/min) as well as during rapid changes in arterial blood pressure induced by sequential boluses of nitroprusside and phenylephrine (Modified Oxford technique). Integrated baroreflex sensitivities for MSNA and HR were analyzed using the slopes of the linear portions of the MSNA‐diastolic blood pressure and RRI‐systolic blood pressure relationships, respectively. When individual cardiac baroreflex sensitivity was compared to sympathetic baroreflex sensitivity, no correlation (R2=0.0003) was found. These results indicate that although both cardiac and sympathetic efferents function in baroreflex control of the autonomic system, there does not appear to be a direct correlation in their function within healthy normotensive individuals.

Research Support: NIH HL083947 and Swedish Medical Council grant 12170
Early‐staged electro‐cardiac conduction in larvae and embryonic zebrafish
Authors: YU FEI , Wolla Christopher David , Chen Jaunian , Hsiai Tzung
Published: June 2021

Zebrafish (Danio rerio) is an emerging model organism for pharmacological studies, tissue engineering, and genetically modified cardiomyocytes. However, early‐staged cardiac function in zebrafish has not been well understood. We proposed to monitor electrocardiography (ECG) in zebrafish at embryonic and larva stage. Larvae were sedated with 0.01% tricane methanesulfonate and placed in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, and 0.16 mM MgSO4) under microscope. A 5μm micropipette filled with 300% Danieau solution were in direct contact with the larva heart for voltage signal recording, and a second micropipette electrode was connected to the ground. Signal processing and wavelet transform enhanced the signal‐to‐noise ratios. ECG signals displaying distinct P wave and QRS complex were acquired from 7 days post‐fertilization (dpf) to 42 dpf. Larva ECG patterns started to transform to the adult features at 35 dpf. When the larvae were treated with Amiodarone (1μM), a Class III anti‐arrhythmic potassium channel blocker, single‐peak QRS complex degenerated into multiple‐peak and wide QRS duration. Thus, we provide a unique in vivo zebrafish model to assess developmental‐staged cardiac conduction phenotype with implication for high throughput research.

Research supported by NHLB 083015 and NHLBI 068689.
Increasing plasma viscosity enhances left ventricular performance in acute moderate hemodilution
Authors: Chatpun Surapong , Tsai Amy G. , Intaglietta Marcos
Published: June 2021

This study investigates how increased plasma viscosity affects cardiac performance during acute moderate hemodilution. Twelve anesthetized hamsters were hemodiluted by 40% of estimated blood volume with plasma expanders. Experimental groups were identified according to the plasma expander viscosity: high viscosity plasma expander (HVPE, 6% dextran 2000 kDa, 6.34 cP) and low viscosity plasma expander (LVPE, 2% dextran 2000 kDa, 2.23 cP). Cardiac function indices were assessed using a miniaturized pressure‐volume conductance catheter. Hemodilution with LVPE reduced whole blood viscosity by 40%, and maintained plasma viscosity relative to baseline. Hemodilution with HVPE reduced blood viscosity to 77% of the baseline and increased plasma viscosity by 1.9 folds relative to baseline. After hemodilution with HVPE, cardiac output significantly increase 24% higher than baseline (p<0.05) whereas it was unchanged after hemodilution with LVPE. Stroke work and maximum rate of pressure change after hemodilution with HVPE were 36% and 20% higher than after hemodilution with LVPE (p<0.05) respectively. These results indicate that increasing plasma viscosity is beneficial in enhancing cardiac function during a moderate isovolemic hemodilution.
The functional differences between biventricular (BiV) endocardial and epicardial pacing in a dyssynchronous left ventricle are dependent on atrioventricular (AV) delay
Authors: Howard Elliot Jacob , Covell James W , Mulligan Lawrence J , Omens Jeffrey H , McCulloch Andrew D , Kerckhoffs Roy C.P.
Published: June 2021

Biventricular (BiV) pacing is used to reduce electromechanical dyssynchrony in patients with left bundle branch block (LBBB). LV lead placement is limited by the patient's anatomy and condition, yielding controversy over selecting an optimal pacing site. We hypothesized that the addition of a LV stimulus that restores transmural activation sequence at late‐activated regions is an important determinant of improved LV function.

To test this hypothesis, dyssynchrony (simLBBB) was produced in open‐chest dogs (n=6). We compared the effects of an additional LV stimulus at late (94.1 ms) activated regions during endocardial (Endo) and epicardial (Epi) pacing at varying atrioventricular (AV) delay. Regional deformation was assessed by biplane cineradiography of implanted beads.

Compared to baseline, simLBBB significantly reduced LV function. Compared to simLBBB, BiV Endo pacing (AV delay of 40 ms) significantly improved systolic function: dPdt max (51%), LVESP (48.8 %), and E33 (0.55). BiV Epi pacing (AV delay of 40 ms) did not differ from simLBBB. At higher AV delays (80–130 ms), BiV Epi pacing significantly improved systolic function, however this improvement was less than BiV Endo pacing.

We conclude that Endo LV pacing has a functional advantage over Epi LV pacing. Endo LV pacing in late‐activated regions may serve to reduce electrical and mechanical dyskinesis, thereby improving ventricular performance.
Effect of relaxation breathing on glycemic response in healthy humans
Authors: Landin Sarah Elizabeth , Covey Stephanie K , Freeman Michelle R , Murphy Shannon K , Ragsdale Frances , Garbrecht Mark R , Wilson Ted
Published: June 2021

Post‐prandial hyperglycemia is a function of glucose absorption, insulin secretion, and insulin sensitivity. A variety of pharmacological interventions improve these parameters. Relaxation breathing is an easily replicated respiratory exercise that may also improve glycemic and insulinemic responsive. In this study healthy subjects were randomized to control breathing (CB; n=13) or relaxation breathing (RB; n=13) groups that received an oral glucose tolerance test (OGTT; 75g/240mL) following a 30 min acclimation period of CB or RB repeated every 10 minutes. Blood glucose was measured at 0 min (pre‐OGTT), and 30, 60, and 90 min post‐OGTT with continued RB (CB: 97±9, 173±30, 159±40 and 137±37; RB: 94±7, 137±29, 166±35, and 130±25mg/dL). RB blood glucose was significantly lower at 30 min (p= 0.004). Plasma insulin for CB and RB at 0, 30, 60 and 90 min was not significantly different, however the insulin AUC for CB and RB was 3155±1272 and 5130±3022 μIU·min/mL (p= 0.098). While pharmaceutical interventions may improve insulin secretion and glycemic control, relaxation breathing could provide a non‐pharmacological approach to improving these parameters in type 2 diabetics. Furthermore, investigators may need to control for respiratory pattern when calculating food glycemic index.
Focal Adhesion Kinase Dynamics under Shear Stress in Live Endothelial Cells Studied with a FRET Biosensor
Authors: Hu Ying‐Li , Li Yi‐Shuan , Wang Yingxiao , Chien Shu
Published: June 2021

Focal adhesion kinase (FAK) plays an important role in integrin‐mediated mechanotransduction signaling events. We transfected endothelial cells (ECs) with a FRET‐based FAK biosensor capable of specifically detecting the phosphorylation of FAK at Tyr‐397, which is reflected in ECFP/YPet emission ratio. The effects of shear stress on FAK activity in live ECs were tracked with high temporal and spatial resolution by recording the FRET responses with a live cell imaging system under flow. The results showed that FAK activity was induced preferentially on the perinuclear and nuclear regions in ECs. To investigate the roles of microtubules and actin filaments in the dynamics of FAK activity, we examined the FRET response in ECs treated with cytochalasin D (CD) or colchicine, respectively. The analysis of FRET responses showed that CD caused an inhibition of FAK activity. In contrast, chochicine caused an increase in FAK activity, particularly in the perinuclear and nuclear regions. These results indicate that the spatial distribution of FAK activity is dependent on the integrity and re‐organization of actin networks, as well as the dynamics of microtubules. The findings suggest that FAK activity may play a significant role in regulating FA formation and cell adhesion to resist cell detachment in ECs subjected to shear stress. This work was supported by NHLBI Research Grants HL‐080518 and HL‐085159 (S.C.).
NO negatively regulates cell surface expression of CD73 in sheared endothelial cells
Authors: Fan Fan , Ohta Masanori , Nandedkar Sandhya , Li Weidong , Jo Hanjoong , Miura Hiroto
Published: June 2021

Both NO and adenosine produced from eNOS and cell surface CD73 in endothelial cells (EC), respectively, are potent vasodilator and anti‐inflammatory molecules. Adenosine increases eNOS expression and activity in EC. We examined if NO regulates cell surface expression of CD73 in EC under shear stress.

In human coronary artery EC, 24‐hour exposure to laminar shear stress (LS; 15 dyne/cm2) increased CD73 mRNA expression (2.1±0.3‐fold vs. static condition, p<0.05, n=10–17), whereas oscillatory shear (OS; ± 5 dyne/cm2) had no effect (0.9±0.2 fold, n=6). However, there was no difference in total CD73 protein levels between LS and OS, since LS caused cleavage of CD73 into the medium (static 22±0.3 and LS 37±0.2‐fold increase vs. fresh medium density, n=4) leading to less surface CD73 expression. Addition of an NO donor DETA‐NONOate abolished surface CD73. This was consistent with less surface expression of CD73 in LS‐exposed regions of mouse aortas than in OS‐regions. On the other hand, an NOS inhibitor LNAME augmented the mRNA up‐regulation (3.7±1.1 fold, n=5) and total and surface protein expressions in LS‐exposed EC. This was consistent with increased CD73 protein expression in eNOS−/− mouse aortas.

These results indicate that NO negatively regulates EC surface expression of CD73 by down‐regulating the mRNA expression and by facilitating cleavage of surface CD73 protein under LS.
Expression of iNOS is reduced by an inhibitor of fatty acyl CoA synthetase.
Authors: Weis Margaret Teresa , Maliha George , Chen Xiong
Published: June 2021

Objective

To test the hypothesis that fatty acyl CoA derivatives of fatty acids modulate cytokine stimulated iNOS expression in C6 astrocytes.

Rationale

Triacsin C, a fungal metabolite, inhibits fatty acyl CoA synthetase in many cell types. The compound inhibits fatty acid induced iNOS expression and apoptosis in isolated islets of Langerhans (Shimabukuro et al 1996), suggesting that fatty acyl CoA derivatives regulate iNOS expression.

Methods

C6 cells were stimulated with a cytokine mix consisting of TNFα (60 ng/mL), ILβ (2 ng/mL) and IFNγ (100 units/mL) in the presence or absence of triacsin C. Expression of iNOS was estimated by measuring iNOS protein by immunoblotting. In vivo iNOS activity was estimated by measuring media nitrite (Greiss reaction) and in vitro iNOS activity was measured by conversion of [14C]arginine to [14C]citrulline in the absence of added calcium.

Results

In C6 cell homogenates, triacin C inhibited FACS activity in a concentration dependent manner, ranging from 31.9 ± 1.5% at 300 nM to 71.1 ± 3.8% at 5 μM. The effect of 5 μM triacsin C on cytokine‐stimulated media nitrite, catalytic activity and immunoreactive protein were measured at 18 and 24 hours:

% of Cytokine Stimulated Activity

Media Nitrite
Catalytic Activity
Immunoreactivity

18 hours
62.5 ± 6.9%
103 ± 11.1%
25.1 ± 13.2%

24 hours
80.6 ± 1.39%*
373 ± 72%*
36.6 ± 5.81%

= p< 0.05 as compared to 18 hours by Student's ttest; n=3 independent experiments for each parameter, each done in duplicate or triplicate.

Conclusions

These results suggest that triacsin C inhibits cytokine stimulated NO formation, and that iNOS protein is significantly less in triacsin C treated cells. Surprisingly, not all of the immunoreactive iNOS protein seems to be catalytically active. The data suggest that triacsin C may delay cytokine‐stimulated expression of iNOS and thus its overall activity in C6 cells.
Diesel exhaust particle exposure exacerbates contractile dysfunction in diabetic‐like cardiomyocytes
Authors: Youtz Dane Juel , Zuo Li , Macmillan Andrew , Rodeman James , Wold Loren Eugene
Published: June 2021

Diesel particulate matter (DEP) pollution and diabetes mellitus (DM) are two key contributors to heart disease in modern cities. The purpose of the present study was to evaluate contractile function, calcium signaling and reactive oxygen species (ROS) formation in adult rat ventricular myocytes exposed to DEP (1 μM) and/or DM simulation media (HG, 25.5mM glucose) overnight. Ventricular myocytes were isolated from adult male Sprague‐Dawley rats and sarcomeric contractile properties were evaluated using an IonOptix Field‐Stimulator system, including: peak shortening normalized to baseline (PS), maximal velocities of shortening/relengthening (±dL/dt), time‐to‐90% peak (TPS90,), time‐to‐ 90% relengthening (TR90), change in fura fluorescence intensity (FFI) and fluorescence decay rate (tau). Intracellular ROS formation was assessed using dihydroethidium. We found that DEP exposure significantly decreased PS, ±dL/dt and increased time‐to‐ 90% relengthening (TR90), tau and intracellular ROS formation in diabetic‐like cells. Further studies indicated that antioxidant co‐culture (0.25 mM Tiron and 0.5 mM N‐Acetyl‐L‐cysteine) completely restored contractile function in DEP‐treated diabetic‐like myocytes. Our final data confirm that DEP exacerbates myocardial dysfunction in diabetic cardiomyocytes, which is mediated by ROS production.
Inhibition of NADPH oxidase 5 activity by NO
Authors: Qian Jin , Fulton David
Published: June 2021

The NADPH oxidases (Nox) are a family of multi‐subunit enzymes that produce superoxide and other reactive oxygen species (ROS). Nox5 is a calcium‐dependent enzyme that does not depend on accessory subunits for activation and in comparison to other Nox isoforms; the molecular regulation of Nox5 is poorly understood. Recently we and others have documented the expression of Nox5 in human blood vessels and the goal of the current study was to determine whether NO can modulate Nox5 activity. Endogenous NO from iNOS was a potent inhibitor of basal and both ionomycin and PMA‐stimulated activity. This action of iNOS was reversible with chronic, but not acute, exposure to L‐NAME and was due to the diffusion of NO, as iNOS was able to inhibit Nox5 when transfected in separate cells. Nox5 activity was also inhibited by eNOS and PM‐targeted eNOS albeit to a lesser extent. The ability of NO to inhibit Nox5 activity was due to a direct modification of enzyme activity as Nox5 activity was inhibited in a disrupted/cell free enzyme activity assay replete with excess calcium and NADPH. Co‐expression of iNOS and Nox5 did not increase the levels of ONOO‐ or induce the tyrosine nitration of Nox5, excluding a role for peroxynitrite. In contrast, there was evidence for the increased nitrosylation of Nox5 using the biotin‐switch assay. Collectively, these results suggest that endogenous NO can directly nitrosylate and inhibit the activity of Nox5.
The antioxidant role of Prdx6 on acute lung injury
Authors: Wang Yan , Fisher Aron
Published: June 2021

OBJECTION

Established the antioxidant function role of Peroxiredoxin 6 (Prdx6) to acute lung injury in vivo or in vitro.

METHODS

100% O2 exposure or H2O2 treatment was used to induced acute lung injury models on intact mice or isolated lung type 2 cells; mice/cells survival rates or lung lavage analysis was applied for evaluating the common defending effect of Prdx6; Tunel assays observed cell apoptosis; Diphenylphosphine (DPPP) fluorescence was used to probe the degree of cell membrane lipid peroxidation.

RESULTS

Prdx6 knockout (KO) mice showed most susceptibility to 100% O2 exposure with shorter survival time and more severe lung injury; on the contrary, Prdx6 overexpression (Tg) mice presented enhanced defending against oxidative lung injury; under varied doses of 50 to 500uM H2O2 treatment for 1 to 24h, Prdx6 KO mice presented dose‐ and time‐related cell injury by increasing the percentage of apoptotic cells, and Tg Prdx6 mice showed least percentage of apoptotic cells; while, the degree of lung type 2 cell membrane lipid peroxidation was probed most serious remarkable in Prdx6 KO mice compared to WT mice and less least in Tg Prdx6 mice under oxidative stress.

CONCLUDSIONS

Prdx6 plays critical antioxidant function role in defending acute lung injury, which associates with its function of anti‐apoptosis and anti‐lipid peroxidation of cell membrane[HL79063].
Cellular and molecular mechanisms of Hydrogen Peroxide (H2O2)‐induced cell Injury and Barrier Dysfunction in Intact Venules
Authors: Zhou Xueping , He Pingnian
Published: June 2021

Reactive oxygen species (ROS) has been implicated to play important roles in the pathogenesis of cardiovascular diseases, but the underlying mechanisms of ROS‐induced cell injury remain unclear. We previously showed that H2O2 induced delayed but progressive increases in intra‐endothelial [Ca2+] (EC [Ca2+]i), microvessel hydraulic conductivity (Lp), and cell apoptosis in intact venules. This study aims to investigate the roles of H2O2‐induced nitric oxide (NO) production and subsequent formation of reactive nitrogen species in H2O2‐induced Lp increases. NO and EC[Ca2+]i were quantified in DAF‐2 and Fura‐2 loaded vessels and permeability was determined by measuring Lp in individually perfused rat mesenteric venules. Perfusing vessels with H2O2 (10 μM) induced an immediate increase in NO and a plateau reached 9.1±0.4 times that of control at 45 min. The increased NO occurred prior to H2O2‐induced increases in EC [Ca2+]i and not affected by Ca2+influx inhibitor LaCl3. Confocal images revealed extensive tyrosine nitration in vessels exposed to H2O2 for 2h but not in untreated vessels. Blocking H2O2‐induced NO production with L‐NMMA not only abolished H2O2‐induced increases in EC [Ca2+]i and tyrosine nitration, but also prevented the Lp increases. Our results indicate that H2O2 induced NOS activation in ECs via a Ca2+ independent pathway and the excessive NO production and subsequent formation of reactive nitrogen species‐mediated cell injury is the key for H2O2‐induced microvascular barrier dysfunction. Supported by HL56237 and HL084338.