bluuebunny/crossref_metadata_2025_split huggingface dataset:
P66Shc‐induced MicroRNA‐34a Causes Diabetic Endothelial Dysfunction by Downregulating Sirtuin1
Authors: Gabani Mohanad , Li Qiuxia , Kim Young‐Rae , Vikram Ajit , Kumar Santosh , Kassan Modar , Kim Sang Ki , Jacobs Julia , Irani Kaikobad 
Published: November 2023

ObjectiveDiabetes mellitus causes vascular endothelial dysfunction and alters vascular microRNA expression. We investigated if endothelial microRNA‐34a (miR‐34a) leads to diabetic vascular dysfunction by targeting endothelial Sirtuin1 (Sirt1), and asked if the oxidative stress protein p66Shc governs miR‐34a expression in the diabetic endothelium.Approach and ResultsMiR‐34a is upregulated, and Sirt1down‐regulated, in aortic endothelium of db/db and streptozotocin (STZ)‐induced diabetic mice. Systemic administration of miR‐34a inhibitor, or endothelium‐specific knockout of miR‐34a, prevents downregulation of aortic Sirt1 and rescues impaired endothelium‐dependent aortic vasorelaxation induced by diabetes. Moreover, overexpression of Sirt1 mitigates impaired endothelium‐dependent vasorelaxation caused by miR‐34a mimic ex vivo. Systemic infusion of miR‐34a inhibitor or genetic ablation of endothelial miR‐34a prevents downregulation of endothelial Sirt1 by high glucose. MiR‐34a is upregulated, Sirt1 is downregulated, and oxidative stress (H2O2) is induced in endothelial cells incubated with high glucose, or the free fatty acid palmitate in vitro. Increase of H2O2 and induction of endothelial miR‐34a by high glucose or palmitate in vitro is suppressed by knockdown of p66shc. In addition, overexpression of wild‐type but not redox‐deficient p66Shc upregulates miR‐34a in endothelial cells. P66Shc‐stimulated upregulation of endothelial miR‐34a is suppressed by cell‐permeant antioxidants. Finally, mice with global knockdown of p66Shc are protected from diabetes‐induced upregulation of miR‐34a, and downregulation of Sirt1, in the endothelium.ConclusionsThese data show that hyperglycemia and elevated free fatty acids in the diabetic milieu recruit p66Shc to upregulate endothelial miR‐34a via an oxidant‐sensitive mechanism, which leads to endothelial dysfunction by targeting Sirt1.Support or Funding InformationNIH grant T32 HL007344NIH grant T32 HL007121University of Iowa Endowed Professorship in Cardiovascular Medicine
Cytosolic Active Caspase 3 Promotes Endothelial Barrier Integrity after Thrombin Stimulation
Authors: Damarla Mahendra , Servinsky Laura , Carino Kathleen , Suresh Karthik , Johnston Laura , Shimoda Larissa , Hassoun Paul 
Published: November 2023

BackgroundWe have previously shown that MK2 deficiency leads to cytoplasmic sequestration of active caspase 3, prevention of apoptosis and is associated with a preserved endothelial barrier function in response to endotoxin challenge. While caspase 3 is widely described as the executioner of apoptosis, it has numerous cytosolic substrates that are not related to execution of apoptosis. Therefore, we hypothesized that cytosolic caspase 3 promotes endothelial barrier function.MethodsHuman lung micro‐vascular endothelial cells (HMVECs) were exposed to a non‐apoptotic, thrombin, stimulus and endothelial barrier function was assessed using electric cell‐substrate impedance sensing (ECIS). After exposures, a subset of HMVECs were evaluated for actin cytoskeletal rearrangement using phalloidin staining. In addition, HMVEC cell lysates were harvested for protein analyses. Caspase 3 knockdown or inhibition was achieved using RNA interference or the pharmacological inhibitor q‐VD‐OPH, respectively.ResultsExposure of HMVECs to thrombin demonstrated a dose dependent transient decrease in endothelial barrier function as measured by a drop in electrical resistance followed by a rapid recovery on ECIS. Thrombin activation of HMVECs led to cytosolic caspase 3 activation, confirmed by fluorescent microscopy. Inhibition of caspases with q‐VD‐OPH in HMVECs led to a similar drop in thrombin‐induced endothelial barrier function but notably there was no subsequent barrier recovery. The lack of barrier recovery was not a result of endothelial cell death but rather a failure of de‐remodeling of thrombin‐induced actin stress fiber formation. Caspase 3 knockdown in HMVECs also resulted in lack of de‐remodeling after thrombin‐induced actin stress fiber formation.ConclusionTaken together, our results suggest cytosolic active caspase 3 promotes endothelial barrier integrity.
Effect of uremia on downregulation of TLR4 and upregulation of cytokines in endothelial cell
Authors: Rebello Jacqueline Ferritto , Brito Rodrigo Barbosa Oliveira , dos Santos Oliveira Willian Rubens , Ribeiro Gabriel Felix , Grabulosa Caren Cristina , Dias Alana Santos , Dellê Humberto , Dalboni Maria Aparecida 
Published: November 2023

INTRODUCTIONThe main cause of mortality in patients with chronic kidney disease (CKD) are Cardiovascular Diseases (CVD); associated with inflammatory mechanisms, which result in endothelial dysfunction and injury. A deficiency of 25 (OH)D3 and/or 1,25(OH)2D3 together with uremic environment may be responsible by poor outcome and activation of the Toll‐like receptors (TLR). Furthermore, it is known that 1,25(OH)2D3 exerts immunomodulatory function in various cell types; however it is unclear whether supplementation with 25(OH)D3 might modulate inflammatory response in endothelial cells against uremic environment.OBJECTIVEThe aim of this study was to evaluate the effect of 25(OH)D3 on the expression of inflammatory markers and intracellular Vitamin D mechanism in endothelial cells against the uremic environment.MATERIALS AND METHODSEndothelial cells from human umbilical cord (HUVEC) were incubated in the presence or absence of uremic serum treated or not treated with 25‐ (OH) D3 for 24h. After incubation, we investigated the inflammatory mediators: Toll‐like (TLR‐4), ICAM‐I (CD54), HLA‐DR, Oxidative Stress (ROS) and intracellular mechanisms of Vitamin D Receptor (VDR), 1 ‐alpha‐hydroxylase (CYP27) and 24‐hydroxylase (CYP24) by flow cytometry.RESULTSThe uremic serum induced lower expression of TLR‐4 compared with the healthy serum HUVEC. Treatment with 25(OH)D3 had no effect on the modulation of TLR‐4 in these cells. For production of ROS, as expected, there was a higher expression in HUVEC stimulated with uremic serum. However, the treatment with 25(OH)D3 not decreased ROS production in these cells. We did not observe differences in the expression of VDR, CYP24 and CYP27 in any groups. We observe a positive correlation between IL‐10, MCP‐1 and ROS and cathelicidin, IL‐10, TNFα and MCP‐1. There was a negative correlation between TLR‐4 and ROSDISCUSSIONThis study showed that the uremic serum induced a lower expression of TLR‐4, higher expression of cytokines and ROS. Unfortunately, the treatment with 25(OH)D3 was not sufficient to diminish these inflammation mechanisms in HUVEC. Thus, it is necessary future studies to evaluate the impact of time response from 25(OH)D3 or 1,25(OH)2D3 in HUVEC in uremic model.Support or Funding InformationFAPESP 2014/14192‐1Universidade Nove de Julho
TNFα Induces Mitochondrial Fragmentation and Mitochondrial Biogenesis in Human Airway Smooth Muscle Cells
Authors: Kaul Esha Sharika , Delmotte Philippe , Sieck Gary C 
Published: November 2023

Airway inflammation is a key aspect of asthma and is associated with airway smooth muscle (ASM) hyperreactivity (airway narrowing due to increased Ca2+ and force responses to agonist stimulation) as well as airway remodeling (ASM cell proliferation and altering extracellular matrix composition). Proinflammatory cytokines, such as TNF‐α mediate this airway inflammatory response. Previously, we found that the TNF‐α induces an increase ASM force, which is associated with an increase in ATP hydrolysis, imposing a metabolic stress on ASM. Mitochondria play an important role in providing ATP to support contraction in ASM. Mitochondria are dynamically remodelling within ASM cells displaying changing filamentous or fragmented tubular networks. Changing expression of Mitofusin 2 (Mfn2), which is associated with mitochondria fusion, or Dynamin Related Protein 1 (Drp1), which is associated with fission mediate this mitochondrial remodelling. The stressors that induce mitochondrial remodelling in ASM are unknown, but mitochondrial fragmentation may be an initial step in mitochondrial biogenesis to meet increasing energy demand. We hypothesized that human ASM (hASM) cells exposed to TNF‐α will display reduced Mfn2 and increased Drp1 expression resulting in mitochondrial fragmentation and mitochondrial biogenesis. To test our hypothesis, serum‐deprived primary hASM cells were exposed to 20 ng/ml TNF‐α for 24 h after which they were loaded with 500 μM MitoTracker Green to visualize mitochondria and imaged using confocal microscopy. Images were analyzed using MATLAB Image J software to quantify mitochondrial morphometry: form factor and aspect ratio. An increase in form factor and aspect ratio indicates greater mitochondrial filamentous networks. Expression of Mfn2 and Drp1 were determined by Western blot analysis. In hASM exposed to TNF‐α, Mfn2 expression decreased by ~40%, while Drp1 expression increased by ~75% compared to controls. These changes in fusion/fission proteins were associated with mitochondrial fragmentation (reduced form factor and aspect ratio) and an increase in mitochondria number. Our results indicate that hASM cells exposed to TNF‐α display mitochondrial fragmentation due to changes in Mfn2 and Drp1 expression. Mitochondrial fragmentation may be involved in mitochondrial biogenesis in response to increased metabolic demands imposed by airway hyperreactivity.Support or Funding InformationAPS fellowship (EK) and NIH grant HL126451 (GCS)
Differential Effects of Cannabinoid Receptor Stimulation in Smooth Muscle
Authors: Bermudez Alexys M , Visina Jacqueline M , Walker Lori A 
Published: November 2023

The use of cannabis as an herbal medicine can be traced back over 5000 years in China, and modern medicine now has well‐documented evidence for cannabis use for treatment of many illnesses. Medicinal marijuana is currently legal in over half of the states in the U.S., and eight states have passed legislation providing for recreational use. A recent survey shows that marijuana legalization support has increased significantly in 2015, nearly doubling in all age groups when compared to 2001. It is estimated that 8.1 million people in the U.S. use marijuana daily. With the increase in use, however, there is an alarming increase in reports of adverse events following marijuana exposure. A French study recently analyzed 35 reports of “remarkable” cardiovascular complications following marijuana use. The cardiovascular events were categorized into cardiac or extra‐cardiac (i.e. vascular) events: Acute coronary syndrome (ACS) composed 57% of the reports; heart rate disorders 5.7%; cerebral vascular events 8.6%; and peripheral vascular events 28.7%. Another study focused on 124 patients admitted for myocardial infarction concluded that there is a 4.8 fold increase in risk of myocardial infarction in the hour immediately following marijuana exposure. We hypothesized that direct stimulation of cannabinoid receptors on smooth muscle could potentiate contractility of vascular smooth muscle. To explore this hypothesis, we first quantified CB1 and CB2 expression in multiple tissues. Interestingly, we found expression of each receptor in every tissue examined. We used isolated vascular (portal vein) and visceral smooth muscle (ileum) preparations to quantify the effects of cannabinoid receptor stimulation. In visceral smooth muscle, stimulation of both CB1 and CB2 with anandamide (a non‐selective cannabinoid receptor agonist) resulted in marked relaxation of contraction while stimulation of CB1 with NADA had no effect on visceral contractility. Because relaxation was not induced through stimulation of solely CB1, the data suggests that relaxation of the visceral smooth muscle is likely regulated through CB2. Conversely, in vascular smooth muscle, stimulation with either CB1 or CB1/2 specific agonists increased contractility. These data suggest that cannabinoid receptor stimulation has a differential effect on visceral and vascular contractility. Future work will be aimed at elucidating the mechanisms underlying differential action of cannabinoid receptor stimulation and will serve to further our understanding of the increase in vascular events correlating with marijuana use.Support or Funding InformationNIH NHLBI 1R25HL103286
SEX DIFFERENCES IN MECHANISMS OF RESISTANCE VESSEL ENDOTHELIAL DYSFUNCTION DUE TO CARDIOMETABOLIC RISK FACTORS AND THE ROLE OF ENDOTHELIAL MINERALOCORTICOID RECEPTORS
Authors: Davel Ana Paula , Moss Maria Elizabeth , Anwar Imran , Lu Qing , Jaffe Iris Zamir 
Published: November 2023

Women with cardiometabolic risk factors (RF) are more likely than men to develop microvascular disease with associated poor cardiovascular outcomes yet the mechanisms driving these sex differences are unknown. We hypothesized that mineralocorticoid receptors (MR) in endothelial cells (EC) contribute to sex differences in resistance vessel dysfunction in response to RF. Male and female EC‐MR knockout (KO) mice were generated by crossing MRf/f mice with VE‐cadherin Cre+ mice, and compared to MRf/f Cre‐ (MR‐intact) littermates as controls. Mice were fed for 12 weeks with standard chow (control), high‐fat diet (HFD) to induce obesity, or HFD with a single injection with adeno‐associated virus (AAV) expressing mutant hPCSK9 to induce hyperlipidemia. EC‐MR deletion did not affect traditional cardiovascular RF including body weight gain, high total cholesterol levels induced by AAV‐hPCSK9, blood pressure, or fasting glucose. In the absence of RF, vasorelaxation of small mesenteric arteries was not affected by the absence of EC‐MR in either sex. Diet‐induced obesity resulted in mesenteric artery endothelial dysfunction, as measured by impaired acetylcholine(ACh)‐induced relaxation, only in MR‐intact female mice. Endothelial function was impaired in hyperlipidemic mice of both sexes. Interestingly, EC‐MR deletion fully prevented endothelial dysfunction in obese and hyperlipidemic female mice, without affecting vasorelaxation in males. Nitric oxide synthase inhibition with L‐NAME and blockade of endothelium‐derived hyperpolarization (EDH) associated with the endothelial K+ channels SK3 and IK1 using Tram34+apamin revealed that obesity and hyperlipidemia cause resistance vessel endothelial dysfunction in females by reducing the EDH component of relaxation. EC‐MR deletion improved vascular function in females, not by restoring the EDH component but rather by enhancing NO‐dependent relaxation. The molecular mechanisms are under investigation with preliminary data supporting that resistance arteries from obese female mice exhibited lower protein expression of IK1, which was not affected by EC‐MR deletion. These data provide a sex‐specific mechanism for microvessel dysfunction due to cardiometabolic risk factors and suggest the possibility that MR blockade may be beneficial in preventing the female susceptibility to endothelial damage in small arteries induced by obesity and hyperlipidemia and the associated morbidity and mortality.Support or Funding InformationFAPESP 2014/26192‐6 (to APD), NIH/NHLBI R01 HL095590 (to IZJ), and AHA EIA 18290005 (to IZJ)
BK channel β1‐subunit deficiency protects against high fat diet‐induced metabolic disorders in the absence of hypertension and exacerbated vascular fibrosis/remodeling in female mice
Authors: Xu Hui , Fernandes Roxanne , Garver Hannah , Galligan James J , Fink Gregory D 
Published: November 2023

Metabolic disorders reduce the expression and/or increase the degradation of BK channel β1‐subunits and cause vascular BK channel deficiency, which is associated with metabolic vascular dysfunction/remodeling in male mice. The contributions of vascular BK channels to control of blood pressure and vascular function/structure have not been studied in female mice. Since estrogen is a potent BK channel activator that protects against metabolic disorder associated cardiovascular dysfunction in premenopausal women, we anticipated that the metabolic disorder associated vascular dysfunction/remodeling would be exacerbated in female BK channel β1‐subunit knockout (KO) mice. 32 weeks of high fat (HF) feeding of female wild‐type (WT) mice caused obesity, moderate hyperglycemia and hyperinsulinemia, hyperlipidemia, hyperleptinemia, and moderate hyperaldosteronism. Compared with HF‐WT mice, female HF‐BK b1‐KO mice showed delayed development of obesity and body weight gain, less visceral adipose accumulation, lower plasma leptin and cholesterol levels, and more adrenal gland hypertrophy. Contractile responses to norepinephrine were unchanged in pressurized MA in vitro in HF‐WT mice, but the responses were significantly reduced in MA from HF‐BK β1‐KO mice, compared with control diet fed mice. All female BK β1‐KO mice were normotensive but exhibited moderate hyperaldosteronism when blood pressure was measured by telemetry. HF‐WT and all BK β1‐KO mice displayed blunted BK channel function in pressurized MA in vitro. All HF mice did not show significant functional and morphological changes to support the exacerbated arterial remodeling/fibrosis and inflammatory responses in mesentery, heart and kidney. Our studies showed that HF diet promotes vascular BK channel deficiency but does not promote hypertension and vascular remodeling/fibrosis in female mice. The non‐vascular BK b1‐subunit deficiency may involve in protection against high fat diet induced metabolic disorders.Support or Funding InformationSupported by NIH2P01HL070687‐11A1
Relationship between body mass index and endothelial dysfunction in women with and without polycystic ovary syndrome
Authors: Adler Tessa E , Usselman Charlotte W , Anaeto Laura C , Schapiro Rebecca G , Steele Frances E , Toffoloni Nikoletta , Takamata Akira , Taylor Hugh S , Stachenfeld Nina S 
Published: November 2023

The flow‐mediated dilation test is a clinical index of endothelial health. Polycystic ovary syndrome (PCOS) is the most common reproductive endocrinopathy affecting ~1 in 10 women of reproductive age. Women with PCOS are at risk for endothelial dysfunction, so we hypothesized that they would have a blunted flow‐mediated dilation (FMD) response relative to controls. We tested 4 women diagnosed with PCOS according to the Rotterdam criteria (age: 30 (6) y, BMI: 26.7 (8.3) kg/m2, range 21.3–40.0 kg/m2) and 4 similarly aged controls (age: 27 (9) y, BMI: 27.6 (9.2) kg/m2, range 18.9–40.2 kg/m2). To assess FMD in the brachial artery of the right arm, brachial diameter was measured at baseline followed by five minutes of occlusion at suprasystolic pressure, and the hyperemic response was monitored for three minutes. Peak diameter was identified and expressed relative to baseline (%FMD). The FMD response was slightly blunted in women with PCOS (8.00 (3.02)%) relative to controls (9.89 (2.73)%). Linear regression analysis revealed an inverse correlation between %FMD and BMI in control women (r2 = 0.61); however, there was a positive correlation between %FMD and BMI within women with PCOS (r2 =0.55). These data suggest that women with PCOS have impaired endothelial function relative to women without PCOS. Further, it appears that BMI predicts endothelial dysfunction only in healthy women. In women with PCOS, endothelial dysfunction occurs independently of BMI. The pathogenesis of cardiovascular disease risk in PCOS is multifaceted and related to risk factors associated with PCOS, such as hormonal imbalance, sympathetic nervous system activation, insulin resistance and hyperinsulinemia.Support or Funding InformationSupported by The John B. Pierce Laboratory and the American Heart Association (15POST24990003).
Obesity Amplifies the Sympathoexcitatory Response to Insulin in Male, but not Female, Rats: Role of Neuropeptide Y Inputs into the Paraventricular Nucleus
Authors: Shi Zhigang , Cassaglia Priscila A. , Brooks Virginia L. 
Published: November 2023

Obesity in males sensitizes the brain to the sympathoexcitatory effects of leptin. However, whether obesity similarly enhances insulin‐induced increases in sympathetic nerve activity (SNA) is unknown. Moreover, while females are resistant to obesity‐induced hypertension, whether the insulin SNA response is sexually dimorphic in obese animals has not been investigated. Therefore, to test the hypothesis that insulin‐induced SNA responses are exaggerated in obese male, but not female rats, we studied SD rats (~8 wks of age) fed a high fat diet (HFD, 33% kcal fat) for 4–6 wk. Both male and female rats diverged into obesity‐prone (OP; top tertile of weight gain) and obesity resistant (OR; bottom tertile of weight gain) groups. Male and female OP and OR rats, as well as control (CON) rats fed a standard diet, were anesthetized with urethane, instrumented with lumbar nerve electrodes and femoral arterial and venous cannulae. Two hr intracerebroventricular (icv) insulin infusions (100 μU/min) in males increased lumbar SNA (LSNA) significantly more (P<0.05) in OP (380±93 % control; n=5) compared to CON (150±16 % control; n=5) or OR (154±13 % control; n=7) rats. However, in females, icv insulin increased (P<0.05) LSNA in CON (to 171±14 % control; n=4) and OR (to 188±13 % control; n=4) rats, but not in OP (to 101±8 % control; n=4) rats. Infusions of artificial cerebrospinal fluid icv had no effects in any group. These results indicate that obesity amplifies the SNA response to insulin in males, but abolishes the response in females. Insulin increases SNA via release of α‐melanocyte stimulating hormone in the paraventricular nucleus (PVN), and this excitation requires simultaneous suppression of tonic inhibitory Neuropeptide Y (NPY) inputs into the PVN. Obesity has been shown to decrease hypothalamic NPY expression, at least in males. Therefore, we next tested if a reduction of tonic NPY SNA inhibition contributes to enhanced insulin responses in OP males, but if NPY inhibition is maintained in OP females. In males, bilateral PVN nanoinjection of the select NPY Y1 receptor antagonist BIBO3304 (NPY1x; 60 pmol/60 nL) increased (P<0.05) LSNA in CON rats (n=6) to 118±2 % control and in OR rats (n=6) to 115±3 % control, but was ineffective in OP rats (to 105±3 % control; n=6). In contrast, in females, PVN NPY1x increased (P<0.05) LSNA in all groups (to 120±5 % control, CON, n=4; 122±6 % control, OR, n=3; and 118±6 % control, OP, n=3). In conclusion, the increase in LSNA in response to icv insulin is amplified in male obese rats, but is abolished in female obese rats. Obesity‐induced suppression of the tonic sympathoinhibitory effects of PVN NPY in males, but not females, may contribute to this sex difference.Support or Funding InformationNIH HL128181 and HL088552, AHA 12GRNT11550018 and 15POST23040042.