bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of prorenin/renin receptor expression on immune cells in an experimental model of preeclampsia in rats
Authors: Sanchez-Guerrero Erika , Rodriguez-Cortes Octavio , Hernandez-Campos Maria Elena , Anguiano-Robledo Liliana , Lopez-Sanchez Pedro
Published: June 2021

The renin‐angiotensin system (RAS) has become more complex. Renin/Prorenin receptor (RPR) is an important component of the renin‐angiotensin system. Its role during normal pregnancy and pregnancy‐induced hypertension (PIH) is not elucidated, especially in the inflammatory response involved in this pathology. The aim of this work was to determine the presence of prorenin/renin receptor (RPR) on immune cells from spleen during normal and pregnancy‐induced hypertension in rats. We used female Wistar rats (250 ± 20 g): 1) non pregnant rats, 2) pregnant rats, 3) pregnant rats whit subrenal aortic coarctation. We used flow cytometry, inmunoblot and RT‐PCR to determine receptor expression on immune cells from spleen. We found expression of (RPR) in non‐pregnant rats, normal pregnant rats, and preeclamptic rats by flow cytometry and immunoblot. Normal rats and normal pregnant rats expressed similar levels of receptor. Preeclamptic rats, however, expressed this receptor in a lower amount. PCR also showed a similar decrease in receptor expression. Based on these results, we can suggest that RPR is expressed on leukocytes and may play a role in the inflammatory response during pregnancy‐induced hypertension. Supported by SIP 20100012 and 20110124.
Blunting of Endothelium Dependent Dilation in Adipose Tissue Arteries by Tumor Necrosis Factor Alpha is Lost after High Fat Feeding
Authors: McCullagh Joseph P , Henson Grant D. , Donato Anthony J. , Lesniewski Lisa A.
Published: June 2021

Tumor necrosis factor alpha (TNF) is implicated in metabolic and endothelial dysfunction with obesity and aging. Although adipose tissue blood flow is reduced with obesity, the cause and consequences on lipid handling and systemic insulin sensitivity are not known. Impaired endothelial function in the resistance arteries of the white adipose tissue (WAT) per se may play a role. We determined if TNF impairs endothelium dependent dilation (EDD) in WAT resistance arteries of young normal chow (Y NC: 4–7 mo) fed mice and if this effect is lost in arteries from Y high fat (Y HF: 8–10 weeks) and old (O NC: 28–31 mo) fed B6D2F1 mice. Maximal EDD to acetylcholine was lower in WAT arteries from both Y HF (68 ± 8%, P<0.01) and O NC (82 ± 6%, P<0.01) vs Y NC mice (95 ± 2%). Recombinant TNF (1 ng/ml, 60 min) reduced EDD in WAT arteries from Y (69 ± 10%, P<0.01) and O NC (47 ± 4%, P<0.01), but not Y HF (65 ± 0.1%) mice. Endothelium independent dilation to sodium nitroprusside did not differ between groups (ALL P>0.05). Compared to Y NC (9.4 ± 1.1 pg/ml), WAT TNF concentration was increased in Y HF (17 ± 3, P<0.01) but not in O NC (11 ± 2) mice. Although these results suggest that previous exposure to excessive TNF in vivo may be responsible for impaired EDD in WAT arteries after HF feeding but not aging, it remains to be determined if endothelial dysfunction in WAT arteries contributes to diminished WAT blood flow and subsequently to inappropriate lipid storage and systemic metabolic/vascular derangements.

Support: NIA‐AG033196, NIA‐AG033755
Novel aspects of leukocyte trafficking
Authors: gavins felicity , hughes ellen , buckingham julia
Published: June 2021

The inflammatory response to the Toll‐like receptor (TLR) 4 ligand lipopolysaccharide (LPS) involves a sequence of intravascular leukocyte capture, rolling and adhesion, followed by their emigration into surrounding tissue which is accompanied by plasma protein extravasation (PPE). Typically, such events are observed hours after LPS injection, but we have chosen to investigate the earlier stages of leukocyte trafficking during murine experimental endotoxaemia.

Using intravital microscopy of the mesentery, we observed a rapid increase in neutrophil emigration and PPE after i.p. LPS injection, reaching a plateau after 2h. The same response was seen with the TLR2 ligand zymosan, but not the non‐specific neutrophil activator ammonium thioglycolate, implicating TLR‐specific mechanisms in early leukocyte trafficking. LPS‐induced trafficking was associated with activation of peritoneal mast cells, shown by selective uptake of the dye ruthenium red from the superfusion buffer, and was inhibited by pre‐treatment with cromolyn sodium.

To conclude, our data suggest that mast cells play a role in LPS‐induced peritoneal inflammation in terms of leukocyte emigration. Further work is underway to determine the sequential mechanisms of cell types and mediators involved in these hyperacute stages of LPS‐induced inflammation.

Work supported by the BBSRC.
Effects of fluids on the liver microcirculation and cell free DNA in sepsis
Authors: Patrick Amanda L , Dwivedi Dhruva , Liaw Patricia , Fox-Robichaud Alison E
Published: June 2021

Fluid resuscitation is a crucial therapy for sepsis. The release of DNA into the circulation in sepsis may predict mortality. Our objective was to determine if our fluids alter hepatic leukocyte recruitment or the levels of plasma cell free DNA (cfDNA).

Cecal ligation and perforation (CLP) was used to induce sepsis in C57BL/6 mice, and compared to sham‐operated controls. Briefly, the cecum was ligated distally, punctured and returned post fecal extrusion. The right jugular vein was catheterized to deliver Seplyte L (SepL) and Seplyte H (SepH), and with or without albumin. Hepatic leukocyte recruitment was assessed by intravital microscopy and plasma cfDNA measured.

CLP mice had higher leukocyte adhesion in sinusoidal and post‐sinusoidal venules compared to shams in all groups. Post‐sinusoidal adhesion was reduced in CLP mice given colloidal SepL and SepH vs. base solutions (13.6± 0.8 vs. 8.1±1.4 and 13.4±1.1 vs. 7.6 ± 0.6; p < 0.05). Six hrs post‐CLP, cfDNA was higher in septic mice when given SepL (9.2±0.3 vs. 4.9±0.5 μg/mL), SepH (8.6±1.4 vs. 4.9±0.4), SepL albumin (9.4±1.1 vs. 5.3±0.2) and SepH albumin (7.2±0.7 vs. 5.3±0.3) (p < 0.03). There was no difference in cfDNA between solutions.

The addition of a colloid to fluids results in less systemic inflammation in early sepsis as measured by hepatic leukocyte recruitment. The production of cfDNA in early sepsis however is not altered by the presence of colloid.
Thrombotic and thrombolytic factors in human plasma with aging
Authors: Field Kristina A. , Murray Erin Patrick , Field Margaret F.
Published: June 2021

Increasing thrombotic disease is seen in the aging population, with reported differences by sex. This study sought to examine thrombotic and thrombolytic factors in human plasma while controlling for age and sex. Subjects ranged from 25–69 yr of age. Thrombotic factors; Fibrinogen (Fb), Factor VII (FVII), Factor X (FX), Tissue Plasminogen Activator Inhibitor (TPA‐I)) and thrombolytic factors; Tissue Plasminogen Activator (TPA) and Antithrombin III (ATIII) were analyzed using sandwich ELISA. Our results show a significant increase in FVII with age in women, with no difference in males. TPA‐I rises in females at perimenopause and remains elevated in menopause. Fb in males is significantly elevated in youngest and oldest groups. TPA‐I was markedly increased in the youngest males only. FX showed a sharp and linear decrease with age in males but not females. We conclude that the rise in clotting factors, Fb in males and FVII and TPA‐I in females may influence increased thromboembolic disease with aging. FX findings suggest that not all factors that are thrombotic play a role in increased thromboembolic disease with aging. The two thrombolytic factors had no significant variation in any experimental group. This research was supported by the Saint Mary's College of California Faculty‐Alumni Grant, Faculty Development Grant, and the Summer Research Program.
Structural microvascular changes as a consequence of cyclophosphamide induced heart failure in rabbits
Authors: van Lier Monique GJTB , Milstein Dan MJ , Coronel Ruben , Spaan Jos AE , Siebes Maria , van den Wijngaard Jeroen PHM
Published: June 2021

Background

Cytostatic drugs are effective in cancer treatment but some have significant cardiotoxic side effects, including heart failure (HF). The aim of this study was to investigate the effect of high‐dose cyclophosphamide (CTX) on fiber and microvasculature (MV) structure of the heart.

Methods

Eight male New Zealand White rabbits (2.9 ± 0.1 kg) were randomly allocated into two groups receiving either 70 mg/kg of CTX (n=4) or sterile 0.9% NaCl (n=4; control) iv once a week for three weeks. Hearts were harvested one week after the last treatment, weighted, and the coronary arterial tree filled with fluorescent casting resin for structural analysis applying our imaging cryomicrotome.

Results

The heart mass of CTX treated rabbits was significantly lower than normal (13.8 ± 0.4 vs. 17.1 ± 0.5; p = 0.04). All hearts were prepared for imaging; however, currently only 1 control and 1 CTX heart has been imaged. Consistent with the loss in heart mass, the preliminary results from the CTX heart showed ventricular dilatation and severe thinning of the ventricle walls. Heart fibers are normally densely arranged; however, the CTX heart consisted of loosely arranged fibers. Also, large transmural vessels are normally radially oriented, a characteristic lost in the CTX heart.

Conclusions

CTX induces HF resulting in loss of fiber and MV structural organization.

Supported by ZonMw 916.11.171
NG2 inhibition decreases endothelial cell sprouting along venules: A novel in situ angiogenesis assay to investigate multicellular interactions
Authors: Stapor Peter Conrad , Ahsan Taby , Murfee Walter Lee
Published: June 2021

Elucidating the multicellular dynamics associated with angiogenesis requires the ability to examine mechanistic responses over the hierarchy of a microvascular network within a controlled environment. The objective of this study was to establish a simple in situ rat mesentery culture model to investigate pericyte‐endothelial interactions during angiogenesis in intact networks. Adult Wistar rat mesenteric windows were aseptically harvested and cultured in minimum essential media for 3 days. Media was left unstimulated or supplemented with bFGF, VEGF or bFGF + NG2 antibody. In supplement‐free media, viability/cytotoxicity analysis revealed that cells were alive after 3 days of culture. Immunohistochemical labeling with antibodies to PECAM, NG2 and LYVE‐1 identified endothelial cells, pericytes and lymphatic cells, respectively. VEGF and bFGF treatments stimulated approximately a 2‐fold increase in capillary sprouting, quantified as endothelial cell sprouts per venule length. NG2 function‐blocking antibodies inhibited the bFGF‐induced sprouting along venules. The results establish primary culture of the adult rat mesentery as a novel in situ angiogenesis model for investigating multicellular angiogenic mechanisms across intact microvascular networks, and implicate pericyte NG2 expression as a functional regulator of capillary sprouting. Supported by the Board of Regents of the State of Louisiana LEQSF(2009‐12)‐RD‐A‐19.

Grant Funding Source : Supported by the Board of Regents of the State of Louisiana LEQSF(2009‐12)‐RD‐A‐19.
Chronic hindlimb ischemia induces cortical angiogenesis and increased stiffness in the tibia
Authors: Govea Michael , Hazelwood Scott , Cardinal Trevor
Published: June 2021

Patients with peripheral arterial occlusive disease are at increased risk of developing osteoporosis. This correlation suggests that peripheral ischemia may play a causal role in the etiology of osteoporosis, which is typically considered a skeletal disease characterized by an imbalance between bone resorption and bone formation resulting in reduced bone mass. Angiogenesis is required for fracture repair and is thought to be a critical component of bone modeling and remodeling. Thus, conditions that impact angiogenesis could directly impact bone remodeling‐related diseases. To determine if ischemia directly impacts bone mechanical properties and remodeling processes, we measured vascular density and performed 3‐point bending in control tibia at days 7, 14, and 28 following unilateral resection of the femoral‐saphenous artery‐vein pair. Tibial stiffness was unaltered at days 7 and 14, but was significantly increased at day‐28. Immunohistochemical staining with GS1 lectin demonstrated increased vascular density at day‐14, proceeding the increased stiffness, and maintained through day‐28. These data suggest that hypoxia‐induced angiogenesis, secondary to arterial resection, is capable of driving bone remodeling responses and mechanical property changes. Future studies will examine the impact of ischemia on osteocytes and test the causal mechanism of ischemia‐induced changes in bone remodeling.
Accelerated Arteriogenesis In Collateral Arterial Segments Exposed To Flow Reversal After Femoral Arterial Ligation
Authors: Meisner Joshua K , Sumer Suna , Niu Jacqueline , Song Ji , Price Richard J
Published: June 2021

Elevated fluid shear stress is known to be a key stimulus for driving arteriogenesis; however, the role that flow‐reversal (FR) plays in arteriogenesis is largely unknown. We hypothesized that FR in spatially distinct regions of collateral arteries (CAs) can lead to spatially distinct variations in CA growth.

METHODS

Unilateral femoral artery ligation (FAL) was performed in C57Bl6 mice. Gracilis muscles were harvested at 1, 7, 14, and 28 days after FAL and immunolabeled for smooth muscle α‐actin to visualize CAs. As determined by intravital microscopy, FAL increased flow across the length of the primary gracilis CAs with distinct regions of flow reversal (FR) and non‐flow reversal (NFR).

RESULTS

Both FR and NFR CA segments showed significant growth as compared to the unligated (control) leg arterial diameters at days 7, 14, and 28 (p<0.001). However, CA regions experiencing FR showed significantly greater expansion of lumenal diameter (arteriogenesis) compared to the NFR regions at days 7 and 14 (p<0.005). This increase became non‐significant by day 28. At day 7, CD45+ leukocyte density was significantly greater around FR CA segments as compared to NFR segments (6.3±3.9 versus 3.9±2.1 CD45+ cells/collaterals, p<0.05, respectively). Further, pericollateral CD45+ cell density significantly correlated with CA diameter in ligated (R2=0.41, p<0.05), but not in unligated CAs (R2=0.0024, p=0.64).

CONCLUSIONS

These data suggest that there are spatial variations in CA growth, CD45+ cell recruitment, and changes in hemodynamics along the length of a given CA after FAL. Together, our data suggest differences in mechanotransduction along a CA lead to differentialgrowth, with increased leukocyte accumulation as a potential mechanism. Supported by NIH HL074082, NIH R21HL098632, AHA 09PRE2060385, AHA 10GRNT3490001.
High salt diet suppresses the angiogenic competency of bone marrow derived mononuclear cells
Authors: Genthe Jamie R. , Greene Andrew S.
Published: June 2021

Bone marrow derived mononuclear cell (BM‐MC) injection is a potential therapy for inducing revascularization in ischemic tissues, however more research is needed into how different disease states affect BM‐MC function. Previously, we have shown skeletal muscle angiogenesis induced by electrical stimulation (ES) is impaired in high salt diet (HSD) fed Sprague Dawley (SD) rats. In this study we tested the hypothesis that injection of BM‐MCs could normalize the microvessel density in HSD fed rats during ES. Following one week on HSD (4% NaCl), 7–8 week old SD rats received 8 hours per day of ES to their right hind limb for 7 days. One day prior to the onset of stimulation, vehicle or BM‐MCs derived from SD donor rats on HSD or normal salt (NSD) were injected intramuscularly into the stimulated limb or intravenously into the tail vein. Microvessel density in muscles of the stimulated limb, relative to the unstimulated limb, was significantly higher in rats injected with BM‐MCs from a NSD donor as compared to vehicle and BM‐MCs from HSD treated rats. We also determined by flow cytometry that a sub‐set of BM‐MCs that were CD34+ and VEGFR2+ were significantly suppressed in the HSD fed rats. This study suggests that HSD impairs the competency of BM‐MC to augment angiogenesis in skeletal muscle following ES and this may be due to a suppression of CD34+/VEGFR2+ cells in the bone marrow.
Perivascular cell dynamics during wrapping‐and‐tapping anastomosis
Authors: Liao Shan , Bazou Despina , Cheng Gang , Munn Lance L.
Published: June 2021

Rapid blood perfusion is critical for post‐implantation survival of thick, prevascularized grafts or bioartificial tissues. In an earlier study, we showed that implanted vascular networks anastomose with host vessels through a process of “wrapping and tapping” between the engrafted endothelial cells (ECs) and the host vasculature. At the host‐implant interface, implanted ECs wrap around nearby host vessels, cause vessel wall reorganization and displace the underlying host endothelium, diverting blood flow to the implanted vascular network. The process is facilitated by high levels of matrix metalloproteinase‐14 and matrix metalloproteinase‐9 produced by the wrapping ECs.

During this process, most host vessels within the brain and implant are fortified with NG‐2‐positive pericytes. In contrast, desmin‐positive pericytes are associated with mature vessels, but not angiogenic host vessels in the gel. Surprisingly, aSMA‐positive perivascular cells are not only associated with large, mature vessels in the brain, but also with the developing HUVEC plexus and wrapped host segments. Thus, aSMA positive stromal cells are involved in formation of the nascent network and desmin staining is associated with of the maturation of the engraft vessels.
Increased sensitivity to endothelin‐1 in posterior cerebral arteries from obstructive sleep apnea rats
Authors: Durgan David Joseph , Crossland Randy F. , Lloyd Eric E. , Phillips Sharon C. , Marrelli Shawn P. , Bryan Robert
Published: June 2021

Obstructive sleep apnea (OSA) has been identified as a significant risk factor for stroke. However, little is known regarding the effects of OSA on the cerebral circulation. The repetitive airway closures associated with OSA lead to intermittent hypoxia/hypercapnia and reoxygenation, increased negative intrathoracic pressures, and arousals. In order to model the physiological consequences of OSA and examine the affect on cerebral circulation, we have chronically instrumented rats with inflatable endotracheal obstruction devices. Rats underwent 30 apneas/ hour for 8 hours/ day (sleep phase). We observed significant decreases in pO2 and pH accompanied by an increase in pCO2 (p<0.05). Additionally, apneas caused a 5‐fold increase in negative airway pressure (p<0.05). Following 2 weeks of apnea OSA rats exhibited a 24‐hour blood pressure (BP) pattern similar to non‐dipping hypertensives (i.e., BP drops <10% during sleep; p<0.05). Using the pressurized cerebral artery preparation, we observed no difference between OSA and sham‐vessels when endothelin (ET‐1) was administered through the lumen of the vessel. However, 1 month of OSA led to significantly increased ablumenal sensitivity to ET‐1 (p<0.05). These data demonstrate that our novel rat model of OSA exhibits characteristics similar to those observed in OSA patients and results in increased sensitivity of cerebral vessels to ET‐1. Funded by R21HL102778
The effects of melatonin and/or exercise on proliferation of neural stem cell/progenitor cell in focal cerebral ischemic rats
Authors: Lee Minkyung , Lee Seunghoon , Lee Youngjeon , Hong Yonggeun Hong
Published: June 2021

Stroke potently stimulates endogenous neuronal stem cell (ENSC) proliferation and neurogenesis and several factors are released by the ischemic brain. The aim of this study was to determine whether melatonin and/or treadmill exercise could induce proliferation of ENSC, thereby promoting morphological and functional recovery in ischemic brain. Sprague‐Dawley (SD) rats were randomly divided into control, MCAo, MCAo+Exercise (Ex), MCAo+Melatonin (MT), MCAo+Melatonin+Exercise (MT+Ex). Following the MCAo, the 8‐week‐old SD rats were treated twice daily with MT (10 mg/kg) at 7:00 and 19:00, trained twice daily with Ex (20 m/min, 6 days/week) at 17:00 and 22:00. We found that MT+Ex was significantly improved the neurological severity score (p < 0.01), as well as the infarct area (p < 0.01). Dendrite density was noticeably increased in the interventional groups with MT and/or Ex compared to MCAo. We confirmed that MT and Ex were significantly enhanced both Nestin and Foxo 3 mRNA level (p < 0.01). On 3rd, 7th, and 28th day, MT and MT+Ex were significantly increased both DCx and GFAP mRNA level (p < 0.01). In conclusion, we suggest that MT and/or Ex intervention might lead to positive effect on proliferation of ENSCs, thereby promoting morphological reconstruction and subsequently followed by functional recovery in MCAo brain.
Chronic administration of kudzu isoflavones ameliorates impaired glucose and lipid metabolisms in obese mice
Authors: Prasain Jeevan , Peng Ning , Rajbhandri Rajani , Wyss J Michael
Published: June 2021

The incidence of type 2 diabetes and metabolic disease is rapidly increasing, but effective therapies for its treatment have been poorly minimally effective. Kudzu root (Radix pueraria from Pueraria lobota) is widely used in traditional Chinese medicine and is a rich source of both isoflavone O‐ and C‐glucosides, the C‐glucosidepuerarin being the most abundant. In this study, chronic administration of kudzu root extract (8 months) decreased fasting glucose by 20% and improved glucose and insulin tolerance. It also increased plasma isoflavone metabolites more in ob/ob than control mice. Unmetabolized puerarin (~12 ng/gram) was in adipose tissue of treated mice. Lipid analysis by tandem mass spectrometry (MRM) demonstrated that ceramide levels were significantly greater in the adipose tissue of ob/ob vs. control mice, and the increased ceramide concentration was reduced by kudzu treatment. Since ceramide inhibits insulin signaling, these data suggest that puerarin containing kudzu induced decreases in adipocyte ceramide levels may contribute to an improved glucose and insulin sensitivity, suggesting that puerarin may be a beneficial adjuvant for type 2 diabetes and metabolic disease.
Mitochondrial Function and Superoxide Production is Reduced in the Diabetic Kidney and Restored by AMPK Activation
Authors: You Young Hyun , Dugan Laura L , Naviaux Robert K , Sharma Kumar
Published: June 2021

Diabetic microvascular complications are considered to be mediated by a glucosedriven increase of mitochondrial superoxide anions. Here we report that superoxide production was reduced in the type 1 diabetic mouse kidney, as assessed by in vivo real‐time transcutaneous fluorescence, confocal microscopy and electron paramagnetic resonance analysis. Mitochondrial biogenesis was reduced with diabetes while pyruvate dehydrogenase (PDH) was phosphorylated, consistent with an overall reduction in mitochondrial glucose oxidation. Mitochondrial biogenesis, PDH activity and mitochondrial complex activity were rescued by the AMPK activator 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR) in both the streptozotocin (STZ) and Akita (Ins2+/C96Y) mouse models of type 1 diabetes. AICAR‐induced superoxide production in the diabetic kidney coincided with reduction of glomerular matrix and albuminuria. Furthermore, AMPKα2 −/− mice were found to have increased albuminuria and AMPK activity was reduced in human diabetic kidney disease. Taken together, the present results demonstrate that diabetic kidneys have reduced superoxide production, and AMPK activation enhances superoxide production and mitochondrial function while reducing disease activity.

This study was supported by grants from the JDRF (KS and LLD) and the NIH for these studies (U01DK076133 KS, R01DK053867 KS, AG 030320 LLD).
PDGF receptor antagonism with imatinib mesylate improves renal hemodynamics independent of changes in blood pressure in Cyp1a1‐Ren2 transgenic rats with ANG II‐dependent malignant hypertension
Authors: Kwak Min-Young , Howard Catherine G , Mitchell Kenneth D
Published: June 2021

The present study was performed to assess the effects of the PDGF receptor‐related kinase inhibitor, imatinib mesylate, on the renal hemodynamic changes that occur during the development of ANG II‐dependent malignant hypertension in Cyp1a1‐Ren2 transgenic rats. Male Cyp1a1‐Ren2 rats (n=5/group) were induced to develop malignant hypertension by dietary administration of indole‐3‐ carbinol (I3C, 0.3% wt/wt) for 10 days. One group was chronically treated with imatinib (60 mg/kg/d, oral gavage) starting 3 days before initiating I3C induction and maintained on imatinib for the duration of the study. In pentobarbital sodium anesthetized Cyp1a1‐Ren2 rats, mean arterial pressure was not significantly different between the induced group and the imatinib‐treated group (182±9 vs. 180±7 mmHg, NS). Rats treated with imatinib exhibited lower renal vascular resistance (31.5±3.0 vs. 45.2±7.0 mmHg/mL/min.g, P<0.05) and increased renal plasma flow (3.15±0.25 vs. 1.95±0.22 mL/min.g, P<0.01) and glomerular filtration rate (1.10±0.11 vs. 0.81±0.09 mL/min.g, P<0.05) compared with induced rats not treated with imatinib. The present data demonstrate that chronic blockade of PDGF receptor‐related kinase improves renal hemodynamics independent of changes in blood pressure in Cyp1a1‐Ren2 rats with ANG II‐dependent malignant hypertension.

Support: NHLBI, Tulane COBRE in Hypertension and Renal Biology.
Activation of Nrf‐2 Enhances the Function of Human Renal Glomerular Endothelial Cells
Authors: Luo Zaiming , Bhupatkar Jenny , Welch William J. , Wilcox Christopher S.
Published: June 2021

Diabetes causes nitric oxide (NO) deficiency dysfunction and nephropathy. Recently, bardoxolone methyl, an activator of nuclear E2‐related factor 2 (Nrf2)–antioxidant response element (ARE) pathway was shown to increase the GFR in diabetic nephropathy, but the mechanism was not established. We detected 1 to 3 potential AREs in the human dimethylarginine dimethylaminohydrolase (DDAH‐1 and ‐2) gene promoter regions. Since DDAH metabolizes the endogenous NOS inhibitor ADMA and upregulates eNOS, we tested the hypothesis that activation of Nrf‐2 enhances endothelial function. Incubation of human renal glomerular endothelial cells (HRGECs) with the Nrf2 activator, tert‐butylhydroquinone (tBHQ; 5–20uM) dose‐dependently and significantly (P<0.05) increased medium nitrite(69±3.7%), NO (62±17.1%) and DDAH activity (47±8.5%), and increased endothelial eNOS, DDAH‐1 and‐2 protein expression by 43±17.1%, 62±20.9% and 48±17.6% (P<0.05). tBHQ markedly increased Nrf‐2 protein in the cell nuclear fraction. In contrast, siRNA knockdown of Nrf‐2, compared tocontrol siRNA, abolished all these tBHQ‐mediated effects and attenuated basal cell proliferation, nitrite and NO production, DDAH activity, and eNOS and DDAH protein expression. In conclusion, activation of Nrf‐2 enhanced endothelial function by simultaneously increasing expression of eNOS and DDAH, thereby increasing NO generation and activity that leads to endothelial cell proliferation. These actions might explain the beneficial effects of Nrf‐2 activation in human diabetic nephropathy.
Protein O‐GlcNAcylation – A Novel Cell Survival Signal in Cardiac Stem Cells
Authors: Zafir Ayesha , Li Qianhong , Bolli Roberto , Jones Steven P.
Published: June 2021

Background

Clinical trials demonstrate the potential efficacy of stem/progenitor cell therapy in the post‐infarcted heart. Yet, the basic biology of these cells remains largely unexplored. Here, we demonstrate that protein O‐GlcNAcylation exerts a pro‐survival role in cardiac stem cells (CSCs).

Methods/Results

Lin−/c‐kit+ CSCs sorted from the adult mouse heart responded to hypoxia‐reoxygenation by increasing protein OGlcNAcylation in a time‐dependent manner upon reoxygenation, along with significantly increased LDH release and caspase‐3/7 activity. We pharmacologically manipulated O‐GlcNAc levels in CSCs with loss‐ and gain‐of‐function approaches, i.e., inhibition of OGT (adds the O‐GlcNAc modification to proteins) by TT04 (0.001 mM), or inhibition of OGA (removes O‐GlcNAc) by ThG (0.025 mM) and subjected them to 3 h hypoxia, followed by 3 h reoxygenation. Reduction in protein O‐GlcNAcylation (via TT04) significantly exacerbated post‐hypoxic cell death (assessed by LDH release, MTS reduction, PI positivity), whereas augmenting O‐GlcNAc levels (via ThG) enhanced cell survival. Diminished OGlcNAc levels render CSCs more susceptible to the onset of post‐hypoxic apoptotic processes via elevated PARP cleavage by significantly enhanced caspase‐3/7 activation, whereas increasing O‐GlcNAcylation serves as a pre‐emptive pro‐survival signal in CSCs by significantly reducing caspase‐3/7 activity and cleaved PARP.

Conclusions

Protein O‐GlcNAcylation represents a robust pro‐survival signal during hypoxia‐reoxygenation injury in CSCs and could be exploited to enhance cell survival prior to in vivo stem cell transplantation for myocardial infarction.

Financial support: NIH, AHA.

*: p<0.05 vs. Normoxia Veh; #: p<0.05 vs. H/R Veh; n≥5/group

image

*: p<0.05 vs. Normoxia Veh; #: p<0.05 vs. H/R Veh; n≥5/group
Adiponectin receptor 1 involves in regulating bone formation and osteoblast differentiation
Authors: Lin Yuan-Yu , Wu Shinn-Chih , Liu Bing-Hsun , Chen Ching-Yi , Mersmann Harry John , Ding Shih-Torng
Published: June 2021

Osteoblasts and adipocytes can derive from the same progenitor cells in bone marrow. The proportion of osteoblasts and adipocytes are associated with age‐related osteoporosis. To evaluate the roles of adiponectin and its receptor (AdipoR1) in regulating the cell fate between adipocytes and osteoblasts, we used the AdipoR1 transgenic and wild type mice to establish mesenchymal stem cells (MSCs) for in vitro study and the bone physiological relevance of the mice were analyzed by micro‐CT analysis. The bone traits of percent bone volume, trabecular number and trabecular thickness were significantly higher in AdipoR1 transgenic mice compared with wild type mice that have the same pattern of serum osteocalcin (bone formation marker) concentration. During differentiation of osteoblasts, the mRNA expression of msh homeobox 2 gene, a key transcription factor of osteoblast differentiation, of AdipoR1‐tMSCs were greater than that of wild type MSCs. Furthermore, paraffin embedding sections of femur has less trap‐positive staining cells in AdipoR1 transgenic mice. These data suggest that the adiponectin may promote osteogenesis and inhibit osteoclastogenesis through its receptor, AdipoR1. Understanding of such mechanism will facilitate the development of treatments for metabolic‐linked bone diseases.
K‐CL COTRANSPORTER‐2 (KCC2) EXPRESSION IN CHICKEN CARDIOMYOCYTES
Authors: Payne John Alan , Antrobus Shane Patrick , Lytle Christian
Published: June 2021

K‐Cl cotransporter‐2 (KCC2) is widely considered to be expressed only in mature central neurons. Here, we report the novel finding that KCC2 is expressed in chicken cardiomyocytes. Previously, we prepared antibodies against a fusion protein encoded by exons 22–24 of rat KCC2 that broadly recognized KCC2 in vertebrate brains. While these antibodies have never shown reactivity with protein outside the CNS of therian mammals, they have never been tested in other tissues of lower vertebrates. We confirmed in rat that KCC2 was exclusively expressed in brain, but in chicken, we observed expression in both brain and heart. In silico analysis showed that while exon 22 is unique to the KCC2 isoform in therian mammals, it is retained in KCC2's closest paralog, KCC4, of lower vertebrates, including chicken. A blocking peptide was used to eliminate cross‐reactivity with chicken KCC4 protein, but it did not prevent reactivity with chicken heart. Furthermore, real time PCR revealed robust KCC2 transcript expression in chicken brain and heart, but nearly 10 times less KCC4 transcript in both tissues. These data confirm that our KCC2 antibodies are indeed recognizing KCC2 in chicken heart. Immunofluorescence microscopy revealed prominent plasma membrane KCC2 labeling in chicken ventricular myocytes. We hypothesize that KCC is critical for cardiomyocyte Cl− homeostasis, especially under conditions of elevated heart rates.
Acoustic trauma reduces SLC4A11 expression in the mouse cochlea
Authors: Yamomoto Hiroshi , Wilson Teresa , Omelchenko Irina , Zhang Yuan , Nakashima Tsutomu , Shi Xiaorui , Nuttall Alfred L.
Published: June 2021

Rationale

The solute carrier 4A11 (SLC4A11) protein, a plasma membrane bound sodium‐borate co‐transporter, is expressed in many different organs including salivary glands, kidney, cornea and the inner ear where it functions to maintain ion homeostasis. Its functional importance is underscored by the causative role of SLC4A11 gene mutations in several inherited diseases, all of which are characterized by some form of sensorineural defect.

Objective

To compare SLC4A11 gene expression with other members of the SLC4 family in the inner ear, eye and kidney and to examine the affect of acoustic trauma on its expression in the cochlea.

Methods and Results

By quantitative real‐time PCR and immunoblot analysis, SLC4A11 transcript and protein levels in whole cochlea were markedly higher relative to eye and kidney, whereas SLC4A2 and SLC4A7 gene expression was quantitatively comparable among the three organs. Acoustic trauma resulted in a significant decrease in SLC4A11 transcript levels in the cochlear lateral wall. Further studies on cultured endothelial cells with chemical hypoxia mimetics revealed that down‐regulation of SLC4A11 can be mediated by hypoxia inducible factor.

Conclusion

Hypoxia, a major source of damage to cochlear tissues from acoustic trauma, may be the mechanism responsible for the reduced levels of SLC4A11.

Supported by grants 5R01DC000105 (ALN), 1R01DC010844 (XS), and P30DC005983.
NS6180, a new KCa3.1 blocker, inhibits T‐cell activation and dampens inflammation in a rat model of inflammatory bowel disease
Authors: Jenkins David Paul , Strobaek Dorte , Brown David T. , Chiu Peter , Jorgensen Susanne , Demnitz Joachim , Wulff Heike , Christophersen Palle
Published: June 2021

The Ca2+‐activated K+ channel KCa3.1 is important in immune cell activation by providing part of the driving force for the Ca2+ entry leading to proliferation and cytokine production. Using a high‐throughput Ca2+‐activated Tl+‐influx assay in hKCa3.1 expressing HEK293 cells, we identified NS6180 (4‐[[3‐(trifluoromethyl)phenyl]methyl]‐2H‐1,4‐benzothiazin‐3(4H)‐one), a benzothiazinone defining a new class of KCa3.1 blockers. In patch‐clamp experiments NS6180 inhibited KCa3.1 with an IC50 of 11 nM via interaction with T250 and V275, the same pore amino acids mediating sensitivity to TRAM‐34. NS6180 blocked the endogenous KCa3.1 channel in human, mouse and rat erythrocytes with nearly identical potencies (15–20 nM). NS6180 suppressed rat and mouse splenocyte proliferation at submicrolar concentrations, but was without effect on splenocytes from KCa3.1−/− mice. NS6180 inhibited IL‐2, INF‐γ, IL‐4 and TNF‐α production but had no effect on IL‐17. NS6180 (3 and 10 mg/kg bid) inhibited DNBS‐induced colitis in rats as effectively as the benchmark therapeutic sulfasalazine (300 mg/kg). Animals treated with NS6180 showed significant improvements in colon weight, fecal occult blood, stool consistency, and body weight gain. We suggest benzothiazinone‐type KCa3.1 blockers as a novel class of anti‐inflammatory compounds and potential drugs.

Funded by NeuroSearch
Down‐regulation of dysferlin and myoferlin in human airway epithelium: differential effects on cell morphology and adhesion
Authors: Leung Cleo , Shaheen Furquan , Hackett Tillie , Bernatchez Pascal
Published: June 2021

Dysferlin and myoferlin belong to Ferlin protein family known to mediate endomembrane fusion events. Mutations of dysferlin lead to muscular dystrophy. Patients with dysferlinopathy show cardiopulmonary complications, suggesting that dysferlin may have functional roles in the pulmonary system. The high homology of myoferlin to dysferlin proposes that myoferlin may also play a role in lung pathology.

Objectives

To examine dysferlin and myoferlin expression in human lungs and investigate the effects of silencing these genes in human airway epithelial cells (HAEC).

Methods

Dysferlin and myoferlin expression was evaluated by immunohistochemistry in human airway sections and by Western blot analysis in HAEC. Localization of dysfelrin and myoferlin in HAEC was studied using immunofluorescent staining. Effects of dysferlin and myoferlin knockdown on the morphology and adhesion of HAEC were also assessed.

Results

Both dysferlin and myoferlin were present in the epithelium of human airway tissues and cultured HAEC. Dysferlin and myoferlin expression localized at the membrane and cytoplasm of HAEC. Knockdown of myoferlin, but not dysferlin, resulted in cell morphological changes, deficient cell adhesion and decreased the expression of zonular occludens 1 in HAEC.

Conclusion

Presence of dysferlin and myoferlin suggests they play potential roles in the adhesion of human airway epithelial cells.
The ventilatory response to muscle metaboreflex stimulation during concurrent hypercapnia
Authors: Bruce Richard Michael , White Michael John
Published: June 2021

The role of skeletal muscle metaboreceptive afferents in human cardiorespiratory control can be examined using post exercise circulatory occlusion (PECO). We have shown that PECO produces ventilatory responses in humans, but only during concurrent hypercapnia and now aim to determine if this interaction is caused by a central or peripheral mechanism. This was achieved by measuring ventilatory and cardiovascular responses to PECO during either systemic hypercapnia (systemic trial) or during local hypercapnia (trapped in the muscle; local trial) respectively. With Ethical Committee approval, 10 males performed 2 trials. Using a cuff inflated to 200mmHg around the right thigh and the inhalation a normoxic hypercapnic gas mixture, manoeuvres were performed to achieve either a systemic or local hypercapnia. In both trials participants then rested for 2min followed by a 1.5min isometric plantarflexion exercise with subsequent PECO for 3.5min before thigh cuff deflation. We found that PECO maintained blood pressure at exercising levels in both trials, suggesting activation of metaboreceptive afferent but ventilation was only maintained at exercising levels in the systemic trial (+6 l.min‐1). These results suggest that ventilatory responses induced by muscle metaboreceptive afferent feedback during concurrent hypercapnia are unrelated to muscle hypercapnia but a central interaction is indicated.
Autonomic boundary conditions for ventricular fibrillation
Authors: Naggar Isaac , Uchida Sae , Kamran Haroon , Lazar Jason , Stewart Mark
Published: June 2021

We sought to describe conditions under which changes in autonomic activity interacted with changes in oxygen availability to cause ventricular fibrillation (VF) in rats anesthetized with urethane. Parasympathetic activation was achieved by stimulation of efferent vagus nerve segments. Sympathetic activation was accomplished by varying doses of systemic isoproterenol. Hypoxemia was induced by varying the dead space attached to an endotracheal tube. BP, ECG, pulse oximetry, and echocardiograms in M‐mode were used. Ventricular fibrillation could be induced with high‐dose isoproterenol, vagotomy, and moderate hypoxemia (O2 sat 50–60%). Lower doses of isoproterenol or non‐zero vagal tone (simulated with vagus nerve stimulation) led to bradyarrhythmia. Severe hypoxemia (O2 sat <50%) also led to bradyarrhythmia. In addition, the dose of isoproterenol necessary to favor VF (≥16 mg/kg) was greater than the dose necessary to achieve maximal heart rate (≤12 mg/kg). We also examined defibrillation. Given that systemic hypoxemia can contribute to a specific set of autonomic conditions favorable for VF, and bradyarrhythmias result when hypoxemia is more severe or vagal tone is present, we conclude VF is an unlikely outcome of extreme autonomic overactivity.
Poiklocapnic hyperoxic hyperventilation (pHH) precedes onset of CNS oxygen toxicity (CNS‐OT): evidence for the hypothesis that hyperbaric hyperoxia (HBO2) stimulates medullary CO2‐ chemoreceptors and respiration prior to seizure
Authors: Pilla Raffaele , Matott Michael , Landon Carol S. , Dean Jay B.
Published: June 2021

HBO2 stimulates CO2‐chemoreceptors, ventilation, and can cause CNS‐OT (seizures). We hypothesize that a predictable pattern of pHH precedes seizure. 118 male rats were implanted with radio‐transmitters to assess diaphragmatic EMG and EEG. After ≥7 days, a rat was placed in a sealed chamber inside a hyperbaric chamber. The rat chamber (100% O2) and hyperbaric chamber (air) were pressurized to 4 or 5 ATA. Each rat underwent 3 dives, 1 dive/week (n = 9 rats/group). Breathing 5ATA O2 induced seizure in 29.2 ± 4.4min, preceded by an increased tidal volume (VT) and respiratory frequency (fRESP) ~5 and ~8min prior to seizure. Similar results were noted at 4ATA O2: seizures occurred after 96.1 ± 10.2 min preceded by increased VT and fRESP ~8min prior to seizure. We conclude that ventilation is a reliable predictor of an impending HBO2 seizure while breathing poikilocapnic hyperoxia at rest. In addition, the ventilatory response to normobaric hypercapnia (5% CO2 in air) was positively correlated with that of pHH at 4ATA (r2= 0.84) and 5ATA (r2= 0.65), suggesting that HBO2 was stimulating central CO2‐chemoreceptor neurons, as we previously reported in rat brain slice studies (ONR).
Development, testing and therapeutic applications of ketone esters (KE) for CNS oxygen toxicity (CNS‐OT); i.e., hyperbaric oxygen (HBO2)‐induced seizures
Authors: D'Agostino Dominic , Pilla Raffaele , Held Heather , Landon Carol , Ari Csilla , Arnold Patrick , Dean Jay B
Published: June 2021

We previously described the effect of KEs, non‐ionized precursors of ketone bodies, in delaying seizures in rats breathing HBO2 at 5 ATA. We hypothesize that oral administration of specific KEs (esters of acetoacetate) will cause a rapid and sustained ketosis that confers neuroprotection against CNS‐OT in rats. Male rats (n = 53) were implanted with radio‐transmitters to measure diaphragmatic electromyogram (dEMG), ECG and EEG. ≥ 7 days post‐surgery, rats were fasted for 12h and administered intragastrically with 1 of 2 KEs (10g/kg), including R,S‐1,3‐butanediol acetoacetate monoester (mKE) or R,S‐1,3‐butanediol acetoacetate di‐ester (dKE) and placed into a hyperbaric chamber and pressurized to 5 ATA O2. Latency to seizure (LS) was measured from the beginning of maximum level of HBO2 until the onset of increased EEG activity and/or tonic‐clonic twitches. Blood was drawn from 18 animals and levels of glucose, pH, pO2, pCO2, β‐hydroxybutyrate, acetoacetate and acetone were analyzed. KEs caused a rapid and sustained ketosis and delayed LS by ~227% (mKE) and ~574% (dKE) compared to control (water). In conclusion, KEs mimic the mild therapeutic ketosis produced by ketogenic diets and confer a significant neuroprotective effect by delaying CNS‐OT (ONR).
Anoxia‐tolerant Western Painted turtle cortex is also ischemia‐tolerant
Authors: Pamenter Matthew Edward , Hogg David William , Gu Xiang Q , Buck Leslie T , Haddad Gabriel George
Published: June 2021

Anoxic or ischemic insults cause immediate hyper‐excitability and cell death in mammalian neurons. Conversely, in Western painted turtle brain, increased GABAergic inhibition suppresses spontaneous electrical activity during anoxia (i.e. spike arrest), and cell death does not occur. To date, the ischemia‐tolerance of turtle neurons has not been investigated, and so to examine this question we treated turtle cortical sheets with a deleterious in vitro mimic of ischemic mammalian brain. We found that ischemia caused: turtle neuronal membrane potential to depolarize from −92 ± 2 to −28 ± 3 mV for the duration of the stress (20 or 60 mins); EGABA to depolarize from −89 ± 1 to −32 ± 3 mV; and input resistance to decrease from 305 ± 33 to 79 ± 12 MΩ. These results suggest that enhanced activation of inhibitory GABAergic mechanisms may also suppress electrical activity in ischemic cortex. Indeed, ischemic neurons were electrically quiet, action potentials could not be elicited, and all changes were reversed to baseline following 20–40 mins re‐perfusion with normoxic ACSF. Furthermore, following 4‐hrs of ischemia, neurons did not exhibit any apparent damage in viability assays; while at 24‐hrs, only early indicators of apoptosis were present. We conclude that anoxia‐tolerant turtle neurons are also ischemia‐tolerant, and that GABAergic inhibition plays a role in this protection.
Neutrophil depletion reduces edema formation and tissue loss following traumatic brain injury in mice
Authors: Kenne Ellinor , Erlandsson Anna , Lindbom Lennart , Hillered Lars , Clausen Fredrik
Published: June 2021

Brain edema as a result of secondary injury following traumatic brain injury (TBI) is a major clinical concern. Neutrophils are known to cause increased vascular permeability leading to edema formation in peripheral tissue, but their role in the pathology following TBI remains unclear. In this study we used controlled cortical impact (CCI) as a model for TBI and investigated the role of neutrophils in the response to injury. The outcome of mice that were depleted of neutrophils using anti‐Gr‐1 antibody was compared to that in mice with intact neutrophil count. Brain edema formation was assessed by Evan's blue dye extravasation and analysis of brain water content. Immunohistochemistry was used to assess cell death and microglial activation. Lesion volume was measured 7 and 14 days after CCI. Neutrophil‐depletion did not significantly affect Evan's blue extravasation at 24 or 48 hours after CCI. However, neutrophil‐depleted mice exhibited decreased water content both at 24 and 48 hours after CCI indicating reduced edema formation. Additionally, neutropenic mice had a decreased number of activated microglia, and brain tissue loss was attenuated in these mice. Furthermore, the number apoptotic cells 24 h after injury was significantly reduced in neutrophil depleted mice. Our results suggest that neutrophils play a role in the edema formation and contribute to the cell death and tissue loss following TBI.
Role of 20‐HETE in neuronal signaling and contribution to neonatal hypoxic‐ischemic encephalopathy
Authors: Koehler Raymond C , Yang Zeng-Jin , Carter Erin L , Kibler Kathleen K , Kwansa Herman , Roman Richard J , Harder David R
Published: June 2021

20‐HETE serves as an intracellular signaling molecule and contributes to infarction in ischemic stroke, but its role in neuronal signaling and in neonatal global cerebral ischemia has not been well studied. Microdialysis probes were inserted in the putamen of anesthetized piglets and perfused with 20‐HETE. Western blot analysis revealed increased serine phosphorylation on Na,K‐ATPase and NMDA NR1 selectively at PKC‐ but not PKA‐sensitive sites. In piglets subjected to hypoxia‐ischemia (H‐I) by systemic hypoxia followed by asphyxia, increased phosphorylation was present in putamen at 3 h of recovery at both PKC‐ and PKA‐sensitive sites on these same proteins. Injection of the 20‐HETE synthesis inhibitor, HET0016 (1 mg/kg), at 5 min of recovery reduced the phosphorylation selectively at the PKC‐sensitive sites on these proteins, improved recovery of Na,K‐ATPase activity, reduced protein carbonyl and nitrotyrosine immunoreactivity, and reduced phosphorylation of Erk1/2 normally seen at 3 h after H‐I. HET0016 did not affect the recovery of cerebral blood flow. Neurobehavioral deficits were ameliorated by HET0016, and over half of the neurons in putamen were protected at 4 days after H‐I. Thus, 20‐HETE can exert direct effects on key proteins involved in neuronal excitotoxicity and contribute to striatal neurodegeneration in neonatal H‐I. (Supported by NIH grants NS060703 and HL59996.)