bluuebunny/crossref_metadata_2025 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

Objective

Diabetes 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 Results

MiR‐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.

Conclusions

These 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 Information

NIH grant T32 HL007344

NIH grant T32 HL007121

University 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

Background

We 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.

Methods

Human 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.

Results

Exposure 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.

Conclusion

Taken 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

INTRODUCTION

The 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.

OBJECTIVE

The 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 METHODS

Endothelial 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.

RESULTS

The 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 ROS

DISCUSSION

This 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 Information

FAPESP 2014/14192‐1

Universidade 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 Information

APS 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 Information

NIH 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 Information

FAPESP 2014/26192‐6 (to APD), NIH/NHLBI R01 HL095590 (to IZJ), and AHA EIA 18290005 (to IZJ)
Bardet‐Biedl Syndrome 1 (BBS1) protein contributes to vascular endothelial function
Authors: JIANG JINGWEI , Reho John J , Rahmouni Kamal
Published: November 2023

Most cells in the human body possess primary cilium, a tiny antenna‐like projection from the cell surface to the environment. In endothelial cells, the primary cilia function as a sensor of blood flow and sheer stress and contribute to both calcium and nitric oxide signaling. Bardet‐Biedl syndrome (BBS) proteins are associated with cilia‐related structures such as the basal body. At least 21 BBS genes (Bbs1‐Bbs21) have been identified so far. The BBS1 protein is an important member of the BBSome complex which contain seven additional BBS proteins (BBS2, 4, 5, 7, 8, 9, and 18). The BBSome mediates protein trafficking to the cilia membrane and perhaps to other cellular compartments. To investigate the role of the cilia protein and the BBSome in endothelial cell function, we generated mice lacking the Bbs1 gene specifically in endothelial cells by crossing mice carrying floxed alleles of the Bbs1 gene (Bbs1fl/fl) with the endothelial –specific Tie2cre mice. The Bbs1fl/fl/Tie2Cre mice developed normally compared to littermate control mice. We assessed vascular function ex vivo using aortic rings and resistance‐sized mesenteric arteries. Interestingly, we found that loss of the Bbs1 gene leads to endothelial dysfunction. In aortic rings, relaxation responses to acetylcholine were decreased in female Bbs1fl/fl/Tie2Cre mice compared to controls (Max. relaxation: 40.1±7.7% vs 73.6±15.3%, P<0.05). The male Bbs1fl/fl/Tie2Cre mice displayed a slightly decreased response that was not statistically different (Max relaxation: 70.4±0.6% vs 76.5±1.7%). In the resistance‐sized mesenteric arteries, both male and female Bbs1fl/fl/Tie2Cre mice showed significantly decreased in relaxation response to acetylcholine (Max. relaxation: male: 38.3±5.5% vs 67.4±5.5% in controls, P<0.05; female: 27.5±12.9% vs 80.8±11.3% in controls, P<0.05). However, the relaxation responses evoked by sodium nitroprusside were not different in male and female Bbs1fl/fl/Tie2Cre mice in both the aorta and mesenteric arteries, indicative of endothelial but not smooth muscle dysfunction in the endothelium‐specific Bbs1 null mice. Thus, disruption of the Bbs1 gene in endothelial cells leads to vascular endothelial dysfunction. Our studies have identified the BBS1 protein as a novel regulator of vascular endothelial function.
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 Information

Supported 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 Information

Supported 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 Information

NIH HL128181 and HL088552, AHA 12GRNT11550018 and 15POST23040042.
Altered Water Homeostasis in a Novel Non‐human Primate Model of Gestational Hypertension
Authors: Weaver Chelsea C , Rhoads Megan K , Grobe Justin L , Osborn Jeffrey L
Published: November 2023

De novo maternal hypertension, or gestational hypertension (GH), is a common yet poorly understood complication of pregnancy that may precede pre‐eclampsia (PE). PE affects 5–10% of pregnancies worldwide and may contribute to 40–60% of maternal deaths in underdeveloped countries. The only known treatment of PE is delivery of the offspring and placenta. Understanding the pathogenesis of GH and/or PE, as well as impacts on the fetus and newborn, could provide more effective diagnostics and treatments. At present, no animal model of spontaneous GH or PE has been identified. In this study, we report characteristics of GH and possibly PE in the nonhuman primate Chlorocebus aethiops sabaeus or vervet. GH was defined as a systolic blood pressure (SBP) exceeding 140 mmHg in the second trimester of pregnancy. Systolic blood pressures (SBP) were obtained using forearm plethysmography under ketamine sedation (15 mg/kg i.m.). Animals were characterized as normotensive (NT; SBP < 120 mmHg), borderline hypertensive (BHT; SBP ≥ 120 mmHg and < 140 mmHg), hypertensive (HT; SBP ≥ 140 mmHg), or GH (SBP ≥ 140 mmHg during pregnancy and NT either pre‐ or post‐pregnancy). Non‐pregnant SBP for NT was 119 mmHg (n = 1) and for GH was 107.67 ± 11.46 mmHg (mean ± SEM, n = 3). Peri‐pregnancy SBP for NT was 112 mmHg (n = 1) and for GH was 154.67 ± 6.39 mmHg (mean ± SEM, n = 3). Mean birth weights of babies born to pre‐pregnancy NT mothers was 309.51 ± 10.15 g (n = 4), to pre‐pregnancy HT mothers was 289.52 ± 18.35g (n = 3), and to GH mothers was 179.23 ± 49.23 g (mean ± SEM, n = 2). The average slope of growth rates from birth to 7–16.5 weeks of age for babies born to NT mothers was 34.04 ± 2.00 g/wk (n = 4), to HT mothers was 26.95 ± 1.80 g/wk (n = 3), and to GH mothers was 43.59 ± 2.34 g/wk (mean ± SEM, n = 2). Average daily water consumption for non‐pregnant females was 77.92 ± 23.53 ml/day (n=6) and for pregnant females was 210 ± 18.36 ml/day (mean ± SEM, n=5, p<0.05). Urine flow rate for non‐pregnant females was 65.56 ± 10.40 ml/day (n=6) and for pregnant females was 97.33 ± 13.05 ml/day (n=5, p=0.06). These data suggest significant positive water balance during pregnancy of female vervets, similar to the response in human pregnancies. Future studies include examination of renal glomerular structure, urinary protein excretion, characterization of plasma and urine osmolarity, osmolar excretion, plasma levels of human PE biomarkers including copeptin, sFlt‐1 and PlGF, free water clearance and placental transcriptomics to establish these animals as a novel spontaneous model of PE. Due to the shared organ physiology (eg ‐ placental structure) and close genetic homology to humans, identification of GH and possibly PE in C. aethiops (vervet) provides a highly translational physiological and genomic animal model to study these severe known pathologies of pregnancy.

Support or Funding Information

Biomedical Science Research Group, LLC and the Gertrude Flora Ribble Foundation
Dihydrotestosterone Mediates Neuroprotective Effects via Estrogen Receptor Beta Following Brain Ischemia‐Reperfusion Injury in Male Mice
Authors: Shi Samuel , Liu Qiang , Gonzales Rayna J
Published: November 2023

Stroke accounts for almost 6.7 million deaths each year and is the foremost cause of disability worldwide. So far therapeutic options are limited clinically. Experimentally, gonadal sex steroids have been shown to be effective neuroprotectants and part of their neuroprotective actions has been attributed to their anti‐inflammatory properties. We have previously shown that androgens, particularly dihydrotestosterone (DHT), an endogenous and nonaromatizable androgen receptor (AR) agonist, and its endogenous downstream metabolite, 5α‐androstane‐3β,17β‐diol (3β‐diol) elicit anti‐inflammatory effects in human cerebrovascular cells during conditions of hypoxia plus glucose deprivation, an in vitro model of ischemia. Effects of DHT and 3β‐diol were AR independent and estrogen receptor beta (ERβ) dependent. Since inflammation is a critical component in the pathogenesis of stroke and our previous data show that these endogenous androgens elicit anti‐inflammatory actions, we investigated whether DHT or 3β‐diol elicits beneficial effects following cerebral ischemic injury. Adult male mice were subjected to transient focal middle cerebral artery ischemia for 60 min followed reperfusion. A bolus amount of DHT (5 mg/kg), 3β‐diol (5 mg/kg), or vehicle (0.01% etoh) was subcutaneously injected immediately following establishment of reperfusion. Neurodeficits and lesion volume were assessed at 24 and 72 h post reperfusion using 2,3,5‐triphenyltetrazolium chloride histology and magnetic resonance imaging. At both time points post reperfusion, DHT and 3β‐diol reduced lesion volume suggesting protection from ischemic injury in both the striatum and cortex. In addition, we also demonstrated that DHT and 3β‐diol significantly improved neurodeficits with the treated population scoring an average of 4 scores less in the modified neurological severity score at 24 and 72 h time points when compared to vehicle controls. Treatment of DHT in combination with the selective ERβ antagonist, PHTPP, reversed DHTs effect and resulted in a comparable lesion size to the vehicle control group at both 24 and 72 h post injury and during reperfusion. This suggests that the neuroprotection observed in the DHT group is ERβ dependent. In conclusion, our data suggest that endogenous androgens are an important mediators of ischemic damage and promote neuroprotection post cerebral ischemia and reperfusion. Our ongoing studies will determine the specific mechanisms associated with DHT and 3β‐diol and their involvement in selective ERβ activation in attenuating ischemic brain injury.

Support or Funding Information

Valley Research Partnership Program Grant (RG) and American Heart Association Grant 16SDG27250236 (QL)
25 (OH) D3 treatment improves inflammatory pathway on monocytes lineage (U937) in uremic environment
Authors: de Oliveira Brito Rodrigo Barbosa , Rebello Jacqueline Ferritto , dos Santos Oliveira Willian Rubens , Grabulosa Caren Cristina , Matos Yves Siva Teles , Malta Camila Soares , Dellê Humberto , Dalboni Maria Aparecida
Published: November 2023

The chronic kidney disease (CKD) is characterized by an inflammatory state caused by the presence of uremic toxins in plasma of patients. This inflammatory condition is associated with a higher risk for cardiovascular diseases and infections. In CKD there is a deficiency of vitamin 25 (OH) D, which has immunomodulatory properties, and that supplementation of the same can decrease inflammation present in uremia.

The aim of this study was to evaluate the effect of 25 (OH) D of inflammatory pathways such as toll like receptor 4 (TRL4), oxidative stress (ROS) and expression of vitamin D receptor (VDR), 1‐α hydroxylase, 24‐hydroxylase and inflammatory mediators in monocytes in uremic environment.

The monocyte U937 cell line were treated with or without 25 (OH) D 50ng/ml for 24 hours and then they were incubated with 50% serum of healthy patients or uremic patient sera Durantes 24 hours at 37 °C and 5 CO2. The monocytes were characterized by expression of CD14 + cells. The TRL4, VDR, CYP24, Cyp27 and ROS was evaluated by flow cytometry. It was performed in the cell culture supernatant ELISA for IL‐6, TNF‐α, IL‐10, MCP‐1 and cathelicidin.

We observed a high expression of TRL‐4, IL‐6, TNF‐α, IL‐10 cathelicidin and MCP‐1 in monocytes incubated with uremic serum, when compared incubated with healthy serum. Pretreatment with 25 (OH) D were able to reduce the expression TRL4, cathelicidin‐1 and MCP opposite uremic environment. There was no difference in the expression of VDR and CYP27 and CYP24 enzymes.

We conclude that the uremic environment induces inflammation by increasing TRL4 and inflammatory mediators and the vitamin D treatment indicates an improvement in the inflammatory parameters and may result in reduced inflammation in uremic environment.

Support or Funding Information

FAPESP 2014/14192‐1
Up‐regulation of Nox4 expression in DUSP4−/− hearts is a critical factor in modulating cardiovascular function under oxidative stress
Authors: Lamb Joseph P , Barajas‐Espinoza Alma , Basye Ariel , Angelos Mark , Chen Chun‐An
Published: November 2023

Increased production of reactive oxygen species (ROS) and decreased antioxidant activity contributing to the cellular redox imbalance is the primary pathogenic factor of ischemia/reperfusion (I/R) injury and endothelial dysfunction. Redox imbalance leads to cellular dysfunction through protein oxidation, DNA damage, and lipid peroxidation. During I/R in hearts, NADPH oxidases (Noxs) are one of the major sources of ROS generation. The burst of ROS production during reperfusion activates stress‐response elements, including the p38 kinase whose over‐activation is one negative contributor to cardiovascular dysfunction, especially I/R injury. Previously we demonstrated that N‐acetyl cysteine (NAC) pre‐treatment up‐regulates dual specificity phosphatase 4 (DUSP4) expression in endothelial cells, regulating p38 and ERK1/2 activities, thus providing a protective effect against oxidative stress. Here, endothelial cells under hypoxia/reoxygenation (H/R) insult and isolated heart I/R injury were used to investigate the role of DUSP4 on the modulation of ROS generation. DUSP4 gene silencing in endothelial cells augments their sensitivity to H/R‐induced apoptosis (45.81% ± 5.23%). This sensitivity is diminished via the inhibition of p38 activity (total apoptotic cells drop to 17.47% ± 1.45%). DUSP4 gene silencing contributes to the increase in superoxide generation from cells. Isolated Langendorff‐perfused mouse hearts were subjected to global I/R injury. DUSP4−/− hearts had larger infarct size than WT. This increase significantly correlates with reduced functional recovery (assessed by: RPP%, LVDP%, HR%, dP/dtmax, low CF%, and a higher initial LVEDP). The level of Nox4 protein and mRNA expression is up‐regulated in DUSP4−/− hearts. This data strongly suggests that DUSP4 is of critical importance in modulating myocardial function after I/R injury and the reason why DUSP4−/− hearts are more susceptible to I/R injury. Thus, Nox4 modulation by DUSP4 provides a therapeutic target.
Renal Inflammatory Response Mediated by Chronic Pain and Related Stress
Authors: Duong Brittany B. , Carder Misty L. , Crosby Garrett S. , Duric Vanja , Babich Victor , Di Sole Francesca
Published: November 2023

Chronic pain is one the most distressing symptoms among patients with chronic kidney disease (CKD). The prevalence of chronic pain in CKD patients has been associated with development of depressive symptoms, while major depression episodes have been linked with a substantially increased risk of death. These evidences suggest that an effective treatment of chronic pain‐related stress and depression may reduce mortality in people with CKD. Moreover, chronic pain‐related stress can correlate to the development of mood disorders and disease of peripheral organs, such as kidney disease, via chronic systemic inflammation and elevation of proinflammatory cytokines. This study aims to determine whether an immune reaction is the mechanism that links depressive behavior induced by chronic pain and anomalies of kidney function. The neutrophil gelatinase‐associated lipocalin (NGAL) and IL‐18 are early diagnostic inflammatory biomarkers. They accumulate in the kidney in response to inflammation, kidney injury and decreased kidney function. Protein levels of NGAL and IL18 were analyzed by immunocytochemistry in animals exposed to chronic pain that have developed depressive‐like behavioral phenotype. NGAL and IL18 protein levels were significantly increased in rat models of neuropathic (spared nerve injury) and inflammatory (injections of complete Freund's adjuvant) pain when compared with their controls; a prevalent increase in NGAL and IL18 protein levels was measured in the inflammatory pain model (~40% increase in both glomeruli and tubules, quantified using novel MATLAB algorithm). These observations suggest that chronic pain and related stress effects induce renal inflammation and possibly a reduction in kidney function. In summary, this study might support a mechanistic understanding of a bidirectional pathway between chronic pain related‐stress and kidney dysfunction.
Decrease in Inflammation (CRP) and Heart Rate Through Mirthful Laughter
Authors: Bains Gurinder , Berk Lee , Lohman Everett , Daher Noha , Miranda Belinda
Published: November 2023

Introduction

With the growth in life expectancy, there is a need for whole‐person care that enhances Quality of Life. Humor associated mirthful laughter has proven to yield numerous whole person health related benefits such as lowering the stress hormone cortisol and improving vascular function. High sensitivity C Reactive Protein (hs‐CRP) is an inflammatory bio‐marker. There are no known studies that compared humor/laughter and a distress state in acute hs‐CRP blood levels.

Purpose

To evaluate the effect of humor associated mirthful laughter and distress on acute CRP and heart rate (HR) modulation in a humor group vs. a distress group.

Methods

Thirty‐two healthy adults were enrolled (humor group, n = 16; distress group, n = 16). The humor group consisted of 8 males and 8 females with a mean age of 26.4 ± 4.4 years. The distress group consisted of 8 males and 8 females with a mean age of 27.1 ± 3.0 years. The hs‐CRP levels were obtained through a finger stick blood test. The hs‐CRP levels were collected at three time points; immediately after signing the informed consent, immediately after watching a 20‐minute humorous video or distressing video, and 30 minutes after the second hs‐CRP test. HR was taken immediately before the first hs‐CRP sample and then five minutes after the first, second, and third hs‐CRP measurement. The humor group self‐selected a humor video of either 20 minutes of Russell Peters stand‐up comedy or a montage of America's Funniest Home Videos. The distress group watched the first 20 minutes of the movie Saving Private Ryan.

Results

There were no significant differences in mean CRP levels and HR over time (p=.24 and p=.06 respectively). For the humor group, CRP levels decreased from 1.65 ± 1.25 mg/L (baseline) to 1.42 ± 1.03 mg/L (post humor); p=.025. For the distress group, there was no significant change in CRP levels for the same time period. For the humor group, HR decreased from 79.5 ± 14.1 bpm (baseline) to 73.4 ± 10.2 (5 minutes post humor) and to 71.2 ± 10.6 bpm (35 minutes post humor); p=.01 and .001 respectively. For the distress group, however, there was no significant change in HR for the same time period.

Conclusion

The health benefits of humor associated mirthful laughter showed a decrease in inflammation (hs‐CRP) and HR and thus, is more beneficial than being in a state of distress. Our pilot study shows potential rehabilitative benefits that can be integrated to whole person wellness platforms. Results indicate that humor and laughter can provide essential components in improving Quality of Life. Further research is needed to expand these positive findings.

Support or Funding Information

Loma Linda University School of Allied Health
Hypoxia/re‐oxygenation increases the response of afferent arterioles to angiotensin II ‐ Investigation in mouse kidney slices
Authors: Pahlitzsch Tamara , Braun Diana , Ludwig Marion , Persson Pontus B. , Patzak Andreas
Published: November 2023

Hypoxia/re‐oxygenation (H/R) plays an important role in the pathogenesis of acute kidney injury (AKI). However, the influence of H/R on cortical microvessel function, especially that of afferent arterioles, is sparsely investigated. We hypothesize that H/R increases the vasoreactivity to angiotensin II (Ang II), which may promote the development of kidney injury. To test the hypothesis, we applied an innovative in situ model of live kidney slices.

Experiments were performed in acute kidney slices (200 μm) of C57BL/6 mice. The TUNEL assay was used to estimate viability of slices by determining the percentage of apoptotic cells. For functional experiments, kidney slices were situated in a chamber at 37°C. Bath solution was equilibrated with carbogen. The afferent arterioles were visualized by differential interference contrast applied to an inverted microscope. To characterize arteriolar reactivity in this model of kidney slices, Ang II, norepinephrine, endothelin‐1 and ATP were administered. For H/R experiments, the slices were transferred into a hypoxia chamber (1% O2) for 30, 60 or 90 minutes and re‐oxygenated for 10 or 20 minutes.

H/R did not significantly influence the apoptotic cell portion in the slice compared to control conditions. Afferent arterioles responded to the bolus application of Ang II (10−7 mol/l) with a maximum constriction, to 44.5 ± 2.8 % of the initial diameter. Norepinephrine (10−5 mol/l), endothelin‐1 (10−5 mol/l) and ATP (10−5 mol/l) reduced the diameter under control conditions to 50.0 ± 2.2%, 45.3 ± 2.6% and 74.1 ± 1.8, respectively. Inhibition of the NO‐synthesis by L‐NAME (10−4 mol/l) significantly enhanced the vasoconstriction to Ang II (32.3 ± 2.3 %). H/R increased the Ang II response of afferent arterioles compared to control conditions. Longer periods of hypoxia were associated with greater arteriolar vasoconstriction to Ang II (30/20 vs. 90/20: 39.7 ± 4.1% vs. 25.9 ± 2.0 %.). Also, increasing the duration of re‐oxygenation resulted in increased vasoconstriction (90/10 vs. 90/20: 34.7 ± 3.5% vs. 25.9 ± 2.00%).

TUNEL staining and functional testing with vasoactive substances indicates the usefulness of the in situ model for the investigation of afferent arteriolar function. The increased response to Ang II in H/R suggests this effect as one component in the multifactorial genesis of AKI.

Support or Funding Information

German Research Foundation (DFG PA 479/10‐2)
Tempol prevents the enhancement of angiotensin II contractions of afferent arterioles from mouse with reduced renal mass
Authors: Li Lingli , Lai En Yin , Luo Zaiming , Griendling Kathy K , Taylor W. Robert K , Wellsten Anton , Welch William J , Wilcox Christopher S
Published: November 2023

Progression of chronic kidney disease (CKD) is related to reactive oxygen species (ROS), impaired arteriolar myogenic contraction and angiotensin II (Ang II). Hence, we tested the hypothesis that ROS modulate the Ang II contractions of afferent arterioles from mice with reduced renal mass (RRM). C57BL/6 mice were subjected to 5/6 surgical nephrectomy or sham operations and fed a 6% salt diet for 3 months. Alternate groups received 2 mmol ·l−1 tempol in drink water. Ang II contractions were measured directly in afferent arterioles and O2− and H2O2 by fluorescence microscopy during applying 10−6 mol·L−1 of Ang II. Compared to sham, mice with RRM had increased O2− and (45.2 ± 4.9% versus 15.0 ± 3.2%; p<0.01), increased H2O2 (35.5 ± 5.0% versus 5.6 ± 0.4%, p<0.01) and enhanced contractions (−72.1 ± 2.5% versus −45.9 ± 3.6%; p<0.05) with 10−6 M of Ang II. Reduction of arteriolar O2− (3.7 ± 2.4 versus 45.2 ± 4.9%, p<0.005) in p47phox −/− mice with RRM blunted the contractions (−35.3 ± 6.0 versus −72.1 ± 2.5%, p<0.005) independent of Ang II concentrations. In contrast, arteriolar contractions in mice with RRM were blunted at high dose (10−6 mol·L−1) but enhanced at low dose (10−9 mol·L−1) of Ang II by metabolism of H2O2 with bath addition of PEG‐catalase (high dose, −55.1 ± 7.1% versus −72.1 ± 2.5%, p<0.005; low dose, −31.1 ± 7.8% versus 10.1 ± 6.8%, p<0.05) or by transgenic overexpression of catalase in vascular smooth muscle cells (high dose, −52.8 ± 1.8% versus −72.1 ± 2.5%, p<0.005; low dose, −19.2 ± 4.6% versus 10.1 ± 6.8%, p<0.05). Therefore, arteriolar H2O2 had Ang II concentration‐dependent effects that contractions are enhanced at high dose but blunted at low dose. Ang II contractions in POLDIP‐2 +/− mice with RRM that did not show an increase in arteriolar H2O2 were similar to POLDIP‐2 +/− sham mice. Three months administration of tempol normalized ROS generation and contractions. The afferent arterioles in mice with RRM expressed much more mRNAs for p47Phox (155 ± 14% versus 100 ± 3, p<0.05), NOX2 (151 ± 16% versus 100 ± 5, p<0.05), Poldip2 (150 ± 17% versus 100 ± 3%, p<0.05), NOX4 (138 ± 12% versus 100 ± 4%, p<0.05) and AT1R (192 ± 6% versus 100 ± 15%, p<0.005) but no change in AT2R mRNA. We concluded that afferent arterioles from mice with RRM had a ROS and/or AT1R associated and Ang II concentration‐dependent enhancement of arteriolar contraction, which were normalized by tempol.
Surviving low oxygen: exploring the role of AMP‐activated Kinase in anoxia‐tolerance
Authors: Van Carter Amen‐Geb
Published: November 2023

High altitude, stroke and cardiorespiratory disorders are damaging to mammalian tissues in that they cause oxygen (O2) deprivation in organs that are energy‐demanding. The zebrafish model provides unique insight into mechanisms of adaptation to O2 deprivation, as zebrafish embryos can adapt to low O2 by entering a state of developmental arrest that conserves cellular energy (ATP). The zebrafish model may therefore reveal targets for therapeutic treatment of hypoxia‐related injury. The Brewster lab aims to identify the signaling pathways that induce the arrested state in response to anoxia (zero O2).

We hypothesize that AMPK (AMP‐activated kinase) is a key mediator of developmental arrest. AMPK is activated by anoxia and conserves ATP by inhibiting cell cycle progression and protein translation in various contexts. We predict that activation of AMPK under anoxia is necessary for the zebrafish embryo to enter developmental arrest. If our hypothesis is correct, we predict that loss of AMPK function under anoxia will lead to premature death. In our ongoing study, we are testing this prediction by comparing arrest and survival rates of Wt (wild‐type) zebrafish embryos to that of mutant zebrafish embryos that are unable to activate AMPK due to a mutation in LKB1, an upstream activator of AMPK.

In order to test this prediction, we are comparing the survival of Wt and LKB1 mutant zebrafish embryos under anoxia. We report here on preliminary studies assessing the LD‐50, the duration of sustained anoxia at which 50% of embryos survive after reintroduction to normal O2‐levels for 24 hours, on Wt embryos at dome stage. We have found that the LD‐50 for Wt zebrafish embryos at dome stage is between 18 and 24 hours under our environmental conditions. Similar studies using LKB‐1 embryos at the dome stage are underway. We anticipate that these studies may reveal a novel role for AMPK in anoxia‐induced arrest and survival.

Support or Funding Information

Funding: American Physiological Society STRIDE program. Acknowledgments: Ikenna Okafor (Graduate Mentor), Sharlene Brown (Graduate Mentor), Dr. Rachel Brewster (PI)