bluuebunny/crossref_metadata_2025 huggingface dataset:
No extrarenal expression of SGLT2 and sex differences in renal expression
Authors: Rose Michael , Gerasimova Maria , Cunard Robyn , Koepsell Hermann , Sabolic Ivan , Vallon Volker
Published: June 2021

Inhibitors of the sodium glucose cotransporter SGLT2 in the proximal tubule have been developed as new anti‐diabetic drugs. Whether SGLT2 also has extrarenal expression is unclear. Here we analyzed SGLT2 protein and mRNA expression in male and female mice (lung, spleen, brain, cerebellum, eye, intraperitoneal fat, heart, testis, uterus, jejunum, submaxillary gland, liver, skeletal muscle and pancreas), and used wild‐type (WT) kidneys and organs of Sglt2 knockout (−/−) mice as positive and negative controls, respectively. A TaqMan® gene expression assay was performed in a real time PCR system and amplification efficiencies standardized against rpl19. Sglt2 mRNA was consistently expressed in WT kidney (Ct value 23.1±0.1) but not detected in other organs at up to 50 cycles. The SGLT2 antibody recognized a specific band close to the predicted size in kidneys of WT but not Sglt2−/− mice. In none of the extrarenal WT organs did the antibody detect a band that corresponded in size to SGLT2 and was unique to WT vs Sglt2−/− mice. Additional studies revealed a 27% lower renal SGLT2 protein expression in females vs males (n=6/gender; P=0.007) despite a 49% higher Sglt2 mRNA expression in female kidneys (P=0.006). The results argue against the expression of SGLT2 mRNA or protein in the extrarenal organs and point to a significant sex difference in renal SGLT2 protein expression that is not explained by different mRNA levels.
Role of interstitial nodal spaces in the urine concentrating mechanism of the rat kidney
Authors: Gilbert Rebecca L , Pannabecker Thomas L. , Layton Anita T.
Published: June 2021

Recent findings in the upper inner medulla of the rat kidney indicate thin limbs of Henle's loops and vasa recta are arranged in repeating patterns among clusters of collecting ducts (CDs). Within CD clusters an interstitial compartment (interstitial nodal space INS) can be seen in transverse sections of the inner medulla. INSs are delimited by a single CD, one or more ascending thin limbs (ATLs) and ascending vasa recta (AVR). In this study we investigated possible fluid and solute flows among CDs, ATLs and AVR, by examining the spatial interactions of structures that form the INSs. Nephron tubules and vasa recta were identified using immunohistochemistry. Spatial interactions were assessed by quantifying lengths of abutting segments of the rat inner medulla. INS architecture was incorporated into a mathematical model simulating steady‐state solute exchange through INSs at given inner medullary levels. At 400 μm below the outer medullary‐inner medullary border approximately 70% of CD was found to abut the INS. In the INS there are negative fixed charges representing macromolecules, e.g. hyaluronan, balanced by Na+ that may promote water reabsorption from CDs. Model results quantify potential fluid and solute fluxes between INSs, nephrons and vessels, and support the hypothesis that INSs serve as fluid and solute mixing chambers that support the inner medullary osmotic gradient.

NIH: DK083338 and NSF: DMS0701412
Fetal Betamethasone Exposure and Age Influence the Expression of AT7/mas Receptors in the Solitary Tract Nucleus
Authors: Marshall Allyson Catherine , Shaltout Hossam A , Nautiyal Manisha , Chappell Mark C , Diz Debra I
Published: June 2021

Betamethasone (BM) is given to women entering into early preterm labor to decrease infant mortality; however, fetal steroid exposure may lead to elevated mean arterial pressure (MAP) in adolescence. We show elevated MAP and decreased baroreflex sensitivity (BRS) at 6 and 20 months (mo) in sheep exposed in utero to BM is associated with alterations in the renin‐angiotensin system favoring a higher ratio of Ang II to Ang‐(1–7). An attenuated BRS is present at 6 wks, prior to increased MAP, and is associated with lower Ang‐(1–7) tone in the dorsal medulla; Ang‐(1–7) through the mas receptor opposes the actions of Ang II on BRS in this region. To establish whether altered mas receptor in the brain medulla contributes to the loss of Ang‐(1–7) tone, we determined the mas receptor protein expression by Western blot analysis in fetal, 6‐, and 20‐mo sheep. No difference between BM‐exposed and control sheep was evident at the fetal or 6‐mo time points. In contrast, mas receptor expression was significantly lower in 20‐mo BM (0.8 ± 0.13) than in control (1.8 ± 0.28) animals. Immunocytochemical data confirms these findings. While BM‐exposed sheep exhibit functional evidence of lower Ang‐(1–7) tone as early as 6 wks of age, the mechanisms may be age‐dependent with lower mas protein in older sheep suggested as a mechanism for the reduced BRS. Support: HD‐047584, HD‐017644, WFSM Venture Fund, Lois Groskert Heart Fund
Role of angiotensin II (Ang II) in the hypertension and renal hemodynamic changes in rats with an altered nephrogenesis. Age‐ and sex‐dependent differences
Authors: Salazar F. Javier , Reverte Virginia , Tapia Antonio , Gambini Juan , Gimenez Ignacio , Moreno J. Manuel , Llinas M. Teresa
Published: June 2021

The aim was to study the role of Ang II in the hypertension and renal changes induced by a decrease of Ang II effects during nephrogenic period (np). It was also examined whether the blood pressure (BP) and renal changes are age‐ and sex‐dependent. Rats were treated with an AT1 receptor antagonist during np (ARAnp) and experiments performed at 3 and 10 months of age. ARAnp‐treated rats were hypertensive but an age‐dependent rise in BP was only found in male rats. Candesartan treatment led to a fall of BP in ARAnp‐treated rats that was greater (P<0.05) in male than in female rats. An increase in oxidative stress is involved in the Ang II effects, since oxidated proteins are elevated (P<0.05) and pretreatment with tempol reduce (P<0.05) the fall in BP elicited by candesartan in ARAnp‐treated rats. Hypertension was not maintained by an increase in AT1 receptor expression in mesenteric arteries and renal cortex. Renal hemodynamic was significantly deteriorated during ageing in male but not in female ARAnp‐treated rats. Renal hemodynamic response to Ang II was enhanced (P<0.05) in these rats at 3 but not at 10 months of age. These results suggest that oxidative stress mediates the involvement of Ang II in maintaining hypertension and renal changes when nephrogenesis is altered during np, and that this involvement is aging and sex‐dependent but not secondary to an elevation in AT1 receptor expression or renal sensitivity to Ang II effects
Gestational chronic intermittent hypoxia causes asymmetric growth restriction and alters cholesterol homeostasis in the liver of sprague‐dawley rats
Authors: Iqbal Waseem , Barry Eric , Hardy Daniel , Ciriello John
Published: June 2021

This study was done to investigate the effect of chronic intermittent hypoxia (CIH) during gestation on the development of offspring. Female Sprague‐Dawley rats were mated, housed individually, and kept on a standard rat chow diet during the study. Pregnant females (n=8) were exposed daily to CIH (6.5% O2 nadir; 18 cycles/h; 8 h/day) from gestational day‐1 to day‐20. Control (n=6) mothers were exposed similarly to room air. Postnatal day‐1 offspring from CIH mothers showed significantly lower body weights, and lower liver‐to‐body weight ratios. By 12‐wk of age, body weights of growth restricted offspring were significantly greater, as were the liver‐to‐body weight ratios. These offspring also had greater body fat deposition and elevated circulating cholesterol levels at both 6‐wk and 12‐wk of age. Western blot and qPCR analysis showed that CIH offspring had decreased protein and gene expression of markers vital for cholesterol regulation in the liver, including Liver X Receptor (LXR), ABCA1 (cholesterol efflux) and the LDL receptor (cholesterol catabolism). These data show that alteration of the maternal intrauterine environment by gestational CIH has significant consequences on the long‐term development of offspring and increases the disposition of the animals to metabolic diseases in adulthood.

Supported by Heart and Stroke Foundation and Canadian Institutes of Health Research
Inhibitory effect of Poria cocos on high glucose‐induced rat mesangial cell proliferation
Authors: Yoon Jung Joo , Lee Yun Jung , Lee So Min , Lee Yong Pyo , Kim Jin Sook , Kang Dae Gill , Lee Ho Sub
Published: June 2021

Mesangial cell proliferation is correlated closely with progression of renal failure. The purpose of this study was to evaluate that water extract of Poria cocos Wolf (WPC), well‐known medicinal plant, regulates rat mesangial cell proliferation under high glucose condition. [3H]‐thymidine incorporation under high glucose was significantly accelerated, which was inhibited by WPC (1 – 50 ìg/ml) in a dose‐dependent manner. Cell migration and fibronectin mRNA expression also suggest that the anti‐proliferative effect of WPC. Western blot analysis revealed that pretreatment of WPC decreased expressions of cyclins and CDKs and promoted expressions of p21waf1/cip1 and p27kip1. WPC also suppressed high glucose‐induced p38 MAPK and ERK 1/2 phosphorylation. Furthermore, WPC inhibited high glucose‐induced production of DCF‐sensitive intracellular reactive oxygen species (ROS). In conclusion, high glucose promotes mesangial cell proliferation, and that WPC has an inhibitory action through, at least in part, inhibition of cell cycle arrest and activation of anti‐oxidant property. Taken together, these results provide that P. cocos may be a potent regulator of proliferation caused by HG exposure.
Effects of K‐deficient diets ± Na supplementation on Na, K, and H2O transporters’ abundance along the nephron
Authors: Nguyen Mien T.X. , Yang Li E. , Fletcher Nicholas K , Lee Donna H. , Silver Randi , McDonough Alicia A.
Published: June 2021

Previous studies of K deficiency compared chow with 2% KCl, 0.74% NaCl (2K1Na ) to K deficient chow: 0.03% KCl, 0.74% NaCl (0K1Na ). We tested whether previously reported responses to 0K diet are due to low electrolytes, rather than hypokalemia. SD rats were fed 6 d with gelled diet prepared from powdered base adjusted to 2K1Na, 0K1Na, and with added NaCl: 2% KCl, 2% NaCl (2K2Na) and 0.03% KCl, 2% NaCl (0K2Na). Abundance was assessed by immunoblot of homogenates, densities normalized to mean of 2K groups.

Results

1) In both 0K groups, plasma K fell to 2.5 mM, urine K fell to < 10 μmol/hr and outer medulla hypertrophied. 2) The only differences seen in both 0K groups are: ↓cortex ENaC and ↑medulla Na,K‐ATPase (NKA). 3) The following changed in 0K1Na but not 0K2Na: ↑cortex gastric H,K‐ATPase (HKA), ↑medulla NHE3, NHE3‐P and ↓AQP2. 4) In 0K2Na but not 0K1Na: cortex SPAK and OSR1 ↑ despite lowest plasma aldo in 0K2Na (172 pg/ml) vs. 0K1Na (413 pg/ml) and 2K2Na (417 pg/ml) and with no changes in NCC‐P or NKCC‐P.

Conclusions

Despite profound hypokalemia and low urinary K, previously reported changes with 0K diets (↑NHE3, ↓NKCC, ↓NCC, ↓ROMK, ↑c‐Src, ↑HKA, ↓AQP2) were not observed; some may be secondary to low electrolytes. ↓ENaC can decrease driving force for K secretion, and ↑NKA is secondary to hypertrophy. Changes in transporter distribution and activity appear more important for conserving K than changes in abundance. DK0837854
Irbesartan improves autonomic tone in a mouse model of renovascular arterial hypertension
Authors: Pliquett Rainer U. , Brandes Ralf P
Published: June 2021

Objective

Activation of renin‐angiotensin‐aldsterone and sympathetic nervous system are consequences of renovascular arterial hypertension. Here, in a model of renovascular hypertension, the role of Angiotensin‐II blockade on autonomic tone was assessed.

Methods

C57bl6n mice with implanted iliac‐artery catheters received a renal‐artery clip (2‐kidneys‐1 clip, 2K1C). Sham‐surgery mice served as normal controls. Blood pressure of unrestrained, conscious animals was recorded using telemetry. Animals were randomized to Irbesartan (10 mg/kg/d per orally) or vehicle for 7 days. Cumulative intensity of the low‐frequency (LF) band of systolic blood pressure variability (SBPV) (0.15 – 0.6 Hz) was considered as sympathetic tone, the high‐frequency band (2.5–5 Hz) of heart rate variability as parasympathetic tone. Spontaneous blood‐pressure changes were used for baroreflex‐sensitivity calculation using Hemolab software. One‐way ANOVA, Newman‐ Keul test were used to assess differences. P<0.05 was deemed significant.

Results

Therapeutic‐dose Irbesartan did not lower blood pressure in the 2K1C model of arterial hypertension (Figure). However, both baroreflex and parasympathetic tone were significantly augmented by Irbesartan. Lastly, the difference of sympathetic tone between 2K1C and normal group partially vanished. Selective blockade of sympathetic tone by Metoprolol and, likewise, parasympathetic tone by Atropine confirmed the changes induced by Irbesartan on either component of autonomic nervous system (data not shown). As a mechanism, baroreflex control of heart rate was shown to be improved by Irbesartan.

Conclusion

Angiotensin‐receptor blocker partially normalizes both baroreflex function and parasympathetic tone in a renovascular model of arterial hypertension.
Effect of OVLT lesion on AngII‐salt hypertension in the rat
Authors: Collister John P , Vieira Alexandre A , Olson Marin K , Nahey David B , Osborn John W
Published: June 2021

We have recently shown that lesion of the organum vasculosum of the lamina terminalis (OVLT) abolishes the hypertensive effects of chronic angiotensin II (AngII) in rats consuming a normal salt diet (0.4% NaCl). While these results support the idea that the OVLT plays an important role in AngII‐induced hypertension, the OVLT has also been suggested as a major sodium sensor in the brain, and the hypertensive effects of AngII are exacerbated by high salt intake. The objective of this study was to investigate the role of the OVLT during AngII‐induced hypertension concurrent with a high salt diet. Male Sprague‐Dawley rats underwent sham (OVLTsham; n = 7) or OVLT lesion (OVLTx; n = 5) and were placed on a high salt (2% NaCl) diet. Mean arterial pressure (MAP) was measured by radio telemetry during 3 control days, 10‐days of AngII infusion (10 ng/kg/min, iv), and a 3‐day recovery period. MAP was not different between groups during the 3 day control period. However, by day 7 of AngII, MAP was higher in sham (158 ± 3 mmHg) compared to OVLTx rats (147 ± 3 mmHg) and remained higher throughout AngII infusion. MAP returned to control levels in both groups during the recovery period. These results suggest that the OVLT contributes to AngII‐salt hypertension in the rat. Supported by HL076312.
Estrogen Deficiency Alters Intestinal Physiology to Promote Bone Loss
Authors: Zhang Jing , Irwin Regina , Raehtz Sandi , Britton Robert , McCabe Laura R
Published: June 2021

Osteoporosis affects over 10 million Americans and another 34 million have osteopenia. Half of menopausal women over the age of 50 will incur an osteoporotic fracture. Although estrogen affects bone cells directly, the mechanisms of postmenopausal bone loss have proven to be complex and multifaceted. Similar to humans, ovariectomized (OVX) estrogen deficient mice display bone loss marked by increased bone resorption and marrow adiposity. Bone density/phenotype is regulated by various factors including the gastrointestinal (GI) system. Little is known about how menopause impacts GI‐bone signaling. In 4‐week post OVX mice we found estrogen deficiency disturbs the balanced gut immune system by up‐regulating inflammatory cytokines such as IL‐1β and IFNγ in jejunum and bone. Concurrently, bone volume is decreased by 40% and correspondingly resorption markers are increased. This is consistent with intestinal inflammation being associated with bone inflammation and loss. We further observed an increase in expression of apoptotic genes in both gut and bone and a corresponding increase in jejunal epithelial cell death as determined by caspase 3 staining. Overall, our studies indicate that estrogen deficiency modifies gut physiology. Understanding how menopause impacts GI‐bone signaling is a critical future area for therapeutic targeting to treat and prevent postmenopausal bone loss.
β1‐adrenergic receptor‐mediated transactivation of epidermal growth factor receptor acutely regulates cardiac gene expression
Authors: Grisanti Laurel A , Talarico Jennifer A , Carter Rhonda L , Radcliffe Scott W , Tilley Douglas G
Published: June 2021

β1‐adrenergic receptor (β1AR)‐mediated transactivation of epidermal growth factor receptor (EGFR) has been shown provide cardioprotection. While the mechanisms responsible for this effect are unknown, we hypothesized that β1AR‐mediated EGFR transactivation could modulate cardiac gene expression. To test this hypothesis, C57BL/6 mice were treated with isoproterenol (ISO) in the presence or absence of an EGFR antagonist (Gefitinib or AG1478). Following 10 minutes of stimulation, phosphorylation of ERK1/2 and Akt were each shown to be increased in response to ISO, and significantly ablated in the presence of AG 1478. Assessment of the subcellular localization of phosphorylated ERK1/2 and Akt in rat neonatal cardiomyocytes in response to β1AR‐mediated EFGR transactivation revealed significant increases in the nucleus, which were ablated by EGFR inhibition. To determine the impact of β1AR‐mediated EGFR transactivation on acute gene expression responses in the heart, mice were treated for 1 hour as described above and real‐time PCR was performed on a panel of EGFR‐regulated pro‐ or anti‐apoptotic genes. Of these, Bcl2l11 expression increased significantly in response to ISO and was attenuated with the addition of gefitinib. Altogether, these results reveal that β1AR‐mediated EGFR transactivation induces differential ERK1/2 and Akt targeting and selective regulation of cardiac gene expression. Funding was provided by the National Institutes of Health (HL‐105414 to DGT).
Interdependence of PPARá/cytochrome P‐450 epoxygenase and adenosine receptors in hypoxia‐induced angiogenesis
Authors: Rizvi Yasmeen , Kasturi Ranganna , Mathew Omana , Myung Choi , Oyekan Adebayo
Published: June 2021

Hypoxia, adenosine and cytochrome P‐450 epoxygenase (CYP epoxygenase) are known to upregulate angiogenesis; however, the role of peroxisome proliferator‐activated receptor alpha (PPARα) in angiogenesis is controversial. Epifluorescent microscopy was used to assess angiogenesis by counting the number of intersegmental (ISV) and dorsal longitudinal anastomotic vessels (DLAV) at 28 hours post‐fertilization (hpf). Under normoxic as well as hypoxic conditions, WY‐14643 (10 μM), an agonist of PPARα, stimulated angiogenesis while MK‐886 (0.5 μM), an antagonist of PPARα, blunted these effects. NECA (10 μM), an agonist of adenosine receptor promoted angiogenesis more effectively under hypoxic conditions as ISV increased by 85% (p<0.001) and DLAV by 100 % (p<0.001) under hypoxic condition. MRS‐1706 (10 nM), a selective A2B antagonist, abolished NECA‐induced increase in angiogenesis. NECA‐ or WY‐14643‐induced angiogenesis was also inhibited by PPOH (25 or 50 μM), an inhibitor of epoxygenase. Moreover, MK‐886 (0.5 μM) inhibited NECA‐induced adenosine receptor mediated angiogenesis and MRS‐1706 (10 nM) inhibited WY‐14643‐ induced PPARα‐mediated angiogenesis under hypoxic conditions. We conclude that hypoxia promoted PPARα as well as adenosine receptor‐mediated angiogenesis and activation of PPARα promoted angiogenesis just as activation of A2B receptors through epoxide‐dependent mechanism.
Perivascular fat impairs contraction in aorta from obese but not lean adult rats
Authors: Watts Stephanie W , McClain Jonathon L , Dorrance Anne M
Published: June 2021

Aortic perivascular fat limits the ability of the aorta to contract. We hypothesized that constriction would be further impaired in rats fed a high fat (HF) diet. Male Sprague Dawley (SD) rats were fed a HF diet from 3–20 weeks of age, control rats received normal chow (n=4–6 in each group). Aortae were removed and isometric force generation was measured in the presence and absence of fat. The HF rats weighed more than the controls (401±9 vs 465±10g, control vs HF p<0.05) and had more abdominal fat (5.6±0.3 vs 16.5±1.2g, control vs HF p<0.05). Surprisingly, the presence of fat in the control rats had no effect on the contraction mediated by phenylephrine (PE) or angiotensin II (Ang II). Fat did cause a marked impairment in the contraction in aorta from HF rats. Interestingly, the clean aorta from the HF rats also exhibited impaired PE and Ang II mediated contraction when compared to clean aorta from control rats. We have shown that fat impairs contraction in aorta from young control chow fed SD rats. The present study suggests that fats ability to limit contraction is reduced with age in control but not obese rats; the mechanisms responsible for this remain to be elucidated.

Maximum contraction (mg increase from baseline)

Contractile Agent
Control
Control + Fat
HF
HF+Fat

PE
1580±210
1595±68
1280±76#
855±59*

Ang II
545±90
533±73
367±80#
157±67*

different from clean aorta from HF rats,

different from clean aorta from control rats
Salt modulates vascular response through cyp‐epoxygenases in the presence of A2A AR
Authors: Pradhan Isha , Mustafa S Jamal , Zeldin Darryl C , Ledent Catherine , Falck J R , Nayeem Mohammed A
Published: June 2021

Recent studies have shown that salt loading enhances adenosine‐induced vascular relaxation (Nayeem et al., 2010). The main goal of this study is to investigate the role of A2A AR in high salt (HS) fed mice using A2AAR−/−and A2AAR+/+ mice. Therefore, we hypothesized that in the presence of A2A AR, HS enhances adenosine‐induced relaxation, whereas in the absence of A2A AR, HS exaggerates contraction. Organ bath and western blot experiments were conducted in 4% NaCl (HS) and 0.18% NaCl (NS) fed A2A AR−/−and A2A AR+/+ mice. Adenosine analog (NECA; 10−6 M) showed relaxation (+12.09 ±4.4 %, p<0.05) in HS‐A2A AR+/+ vs. contraction in both NS‐A2A AR+/+(−32.3 ± 4.9%) and HS‐A2AAR−/−(−70.24 ± 5.61%). CGS‐21680 (A2A AR agonist) enhanced relaxation in HS‐A2A AR+/+ (+25.16 ± 4.70%, P < 0.05) vs. less relaxation in HS‐A2AAR−/−(+8.07 ± 2.36%), and relaxation was blocked by an EET antagonist (14,15‐EEZE ; 10−6 M, −1.08 ± 2.95%). Compared to NS‐A2AAR+/+, HS‐A2AAR+/+ demonstrated up‐regulation of A2AAR, cyp2c29 (30%, 36%, respectively) and down‐regulation of A1AAR, cyp4a (8%, 32%, respectively). Up‐regulation of A1AAR, cyp4a (18%, 80%, respectively) and down‐regulation of cyp2c29 (36%) in HS‐A2AAR−/−vs. HS‐A2AAR+/+ were observed. In conclusion, HS enhances relaxation through cyp2c29 in A2A AR+/+, whereas HS exaggerates contraction via CYP4A in A2AAR−/−mice. WVU‐HSC, NIH: HL 027339 & HL 094447, GM 31278, NIEHS: z01 ES025034.
FORMATION OF POLYMERS INSULIN IN VITRO IN BLOOD HATCHING TIBOLONE IN MENOPAUSAL PATIENTS
Authors: moral-laguna elia e del , Valencia-Hernandez Ignacio , de la Luz Linares-Perez Maria , Rivera-Llano Aidé , Lugo-Martinez Gabriela , Ocharan-Hernandez Maria Esther , Calzada-Mendoza Claudia Camelia
Published: June 2021

OBJECTIVE

Evaluate in vitro the formation of polymers of insulin after blood incubation in menopausal patients treated with tibolone, also quantify oxidative stress markers and apply anthropometry.

METHODS

Were selected 18 menopausal patients 45–50 years of age, whose was treated whit tibolone for three months (2.5mg/day), whose blood was incubated in insulin, underwent same nondenaturing electrophoresis and visualize the polymers of the hormone. Additionally we evaluated markers of oxidative stress (malondialdehyde, total sulfhydryl and carbonyl groups) and anthropometric parameters.

RESULTS

It was observed that blood from all patients before treatment induced the formation of polymers of insulin and after treatment, only 33% remains. The oxidative stress evaluation shows reduction in damage to lipids (malondialdehyde), increased sulfhydryl groups ( they are part of the antioxidant system), but the proteins damage indicator (carbonyls) still unchanged. Regarding anthropometric assessment was observed weight reduction (p = 0.013), BMI (p = 0,035) and glucose (p =. 016).

CONCLUSIONS

Tibolone reduced the oxidized state of the blood and its ability to induce the formation of polymers of insulin, weight and body mass index and blood glucose.
Diosgenin protects doxorubicin‐induced cell apoptosis in macrophages: involvement of MAPK and ATF‐3 pathways
Authors: Park Bongkyun , Park Daesup , Rhee Dong-Kwon , Pyo Suhkneung
Published: June 2021

Doxorubicin (DOX), a derivative of anthracyclines, is a widely used anticancer drug in the treatment of a broad spectrum of cancers. Dox is non‐selective cytotoxic drug with significant side effects on immune cells. We investigated the influence of diosgenin, a precursor of steroid hormones, on apoptosis in mouse peritoneal macrophages. Pretreatment of cells for 1 h with diosgenin (1–5 μg/ml) inhibited DX‐induced cell death in a concentration‐ and time‐dependent manner. Western blot assay showed that diosgenin suppressed caspase‐3 activation and PARP cleavage in doxorubicin‐treated macrophages, suggesting the prevention of doxorubicin‐induced apoptosis. We also found that diosgenin inhibited the down‐regulation of Bcl‐2, whereas the level of Bax and p53 expression was decreased by diosgenin treatment in Dox‐treated macrophages. Diosgenin inhibited Dox‐induced ATF‐3 expression and phosphorylation of p38 and ERK1/2. Furthermore, inhibition of ATF‐3 activity by shRNA suppressed DOX‐induced caspase‐3 activation and apoptosis. These data indicate that protective effect of diosgenin may occur through the regulation of p38, ERK1/2 and ATF‐3 signaling pathways.
Beta‐arrestin2 Contributes to the Development of Opioid‐ Induced Constipation
Authors: Raehal Kirsten M , Bohn Laura M
Published: June 2021

Constipation is a common and problematic side effect associated with opioid therapy. Opioid analgesics such as morphine exert their physiological effects primarily through activation of the mu opioid receptor (MOR), a G protein‐coupled receptor (GPCR). Although opioids are known to decrease gastrointestinal motility by decreasing secretion and muscle contractions, the cellular signaling mechanisms downstream of MOR activation that mediate these physiologies are not completely understood. We have previously shown that morphine‐induced constipation is significantly reduced in βarrestin2 knockout (βarr2‐KO) mice. Moreover, this reduction appears to be due to altered opioid receptor responses in the enteric nervous system, specifically at the level of the colon. Here, we have assessed the contribution of βarrestin2 in mediating morphine‐inhibition of secretion within the colon by assessing changes in nicotinic stimulation of secretion in colonic tissue preparations using Ussing chambers. We find that the βarr2‐KO mice display significantly less morphine‐induced inhibition of secretion compared to WT controls, suggesting that βarrestin2 may facilitate the effects of constipatory effects of morphine in the colon. These results provide further evidence that βarrestin2 is a critical regulator of morphine‐induced constipation. Supported by NIDA DA021952 (KMR), DA14600 and DA18860 (LMB).
Zuntz Revisited: Contributions to Exercise Physiology
Authors: Tipton Charles M.
Published: June 2021

Although Nathan Zuntz (1847–1920) was nominated for the Nobel Prize on three different occasions in subjects central to physiology, he has received minimal recognition by exercise physiologists. Early in his career he became interested in the physiology of performance by both humans and animals when perfecting the equipment and methodology to secure respiratory data. As a consequence, treadmills were developed and climatic chambers constructed. O2 consumption results were obtained from subjects marching, climbing, cycling, swimming and typing. He perfected the use of the Fick Principle and secured cardiac output values from exercising horses. Besides obtaining the work of the heart and aortic blood pressures values from horses, he was able to secure exercise changes in cardiac dimension of humans (via portable x‐ray measurements) when exercising on the treadmill. From his classic marching study, he reported marked increases in RBC and leucocytes; improper clothing coupled with high humidity and air temperatures impaired exercise thermoregulation, and that dehydration during marching must be avoided. Moreover, he opposed withholding water from marching soldiers or workers. He believed contracting muscles utilized carbohydrates (CHO) and fats in proportion to the amount delivered to the tissues; CHO were not converted to fat before utilization; proteins were not substrate with exercise, and that the RQ (RER) value would decrease with prolong exercise. These views were not in agreement with those of Liebig or Chauveau.
Assessment of Nova Biomedical StatStrip® Glucose Meters and Test Strips in Rodent Glucose Studies
Authors: Peterson Richard George , Brockway Robert
Published: June 2021

Preclinical blood glucose testing methods have relied largely on human consumer market technology. The FDA accuracy requirement for consumer meters of +/−20% is not ideal for research. The StatStrip Xpress GLU meter (Nova Biomedical) is only marketed for use in hospital settings. An “investigational use only” meter with a range to 900mg/dL is now available through DSI. The StatStrip system is immune to common interferents such as ascorbic acid and acetaminophen and also measures and corrects for hematocrit levels in the blood; consumer meters do not. The AlphaTrak (Abbott) meter is specifically marketed for preclinical use and has demonstrated higher accuracy in animal models than consumer meters. The present study compares both systems to the model AU480 (Beckman Coulter) clinical analyzer. Two measurements were made with each of the systems on blood samples from both normal and diabetic ZDSD rats (N=98). The results showed the AlphaTrak to have a bias of −9.0% relative to the AU480 across a range of glucose from 100–600mg/dL with a zero crossing of +7.8mg/dL, while the StatStrip had a bias of 3.6% relative to the AU480 with a zero crossing of −10.1mg/dL. The R2 value was slightly better for the StatStrip readings at 0.993 versus 0.966 for the AlphaTrak. Overall, the StatStrip system provided more accurate data than the AlphaTrak system and data nearly equivalent to the clinical analyzer over the range of glucose studied.
Decreased caveolin‐3 and increased GlcNAcation in cardiac myocyte caveolae during diabetes mellitus
Authors: Finley J Cameron , Kidd Michael W , Scott Brian T , Roth David M , Dillmann Wolfgang H , Patel Hemal H
Published: June 2021

Cardiomyopathy that is independent of dysfunctional vasculature occurs in patients with diabetes mellitus (DM), thus suggesting that factors within cardiac myocytes contribute to DM‐associated cardiac remodeling. Caveolin‐3 (Cav‐3), a membrane structural protein, can modulate cardiac remodeling where mice with knockout of Cav‐3 have a cardiomyopathic phenotype and overexpressors are protected. To test the hypothesis that post‐translational modification of caveolar proteins and mitochondrial dysfunction occur in DM, we assessed hearts from control or streptozotocin‐induced DM mice using fractionation/immunoblot analysis, electron microscopy (EM), and mitochondrial function assays. We found that diabetic hearts have reduced Cav‐3 expression and increased O‐GlcNAcation in Cav‐3 enriched membranes. EM revealed that diabetic hearts had disruption of cytoarchitecture with disorganized, rounded mitochondria and fewer caveolar invaginations. Functional studies showed that mitochondrial respiration in DM myocytes is impaired and oxidative stress enhanced. Therapies designed to enhance Cav‐3/caveolae expression thus are potentially novel approaches to limit DM‐associated cardiac remodeling.
Mitochondrial mechanism of right ventricular failure (RVF)
Authors: Qipshidze Natia , Tyagi Neetu , Metreveli Naira , Tyagi Suresh C
Published: June 2021

Sustained pressure overload induces pathological cardiac dysfunction, linked to mitochondrial dysfunction (mitophagy) during RVF. We tested whether mitophagy division inhibitor (Mdivi‐I) mitigates oxidative stress and leads to reverse pre‐established advanced hypertrophy, fibrosis, and RV dysfunction. To verify this, C57 (less), FVB (intermediate), and C3H (most resistant to oxidative stress) strains of mice underwent pulmonary artery constriction (PAC) for 4 weeks with or without the Mdivi‐I (50 mg/kg/day) treatment. After PAC mice developed increased cardiac mass, lowered ejection fraction, triggering oxidative stress, and mitochondrial dysfunction. Mdivi‐I treatment reversed hypertrophy and fibrosis, lowered oxidative stress, and improved chamber and myocyte function, whereas untreated hearts worsened, To further support the claim of mitophagy occurrence during RVF, the levels of LC3A/B and P62 were measured. LC3A/B was increased in RV of PAC mice. Similarly, increased P62 protein level was also observed in RV of PAC mice. Treatment with Mdivi‐I abolished this affect in PAC mice. RV pressure in PAC mice was increase up to 55±3 mmHg, treatment with Mdivi‐I decreased the pressure to 32±4 mmHg. These results suggest that, Mdivi‐I treatment reduces oxidative stress and ameliorates mitochondrial dysfunction that results in protection of RVF against mytophagy during PAH.
AMP‐activated protein kinase activation inhibits human endothelial cell proliferation
Authors: Durante William , Liu Xiao-ming , Yu Yajie , Yates Benjamin , Peyton Kelly J.
Published: June 2021

AMP‐activated protein kinase (AMPK) is a ubiquitously distributed energy sensing enzyme that is activated by changes in cellular metabolism. Recent studies indicate that AMPK influences endothelial cell (EC) function and vascular homeostasis, but the underlying mechanisms are not fully known. In the present study, we investigated whether AMPK regulates EC proliferation. Treatment of cultured human umbilical vein ECs with serum stimulated a time‐dependent increase in cell number that was inhibited by the AMPK activators, 5‐aminoimidazole‐4‐carboxamide‐1‐â‐D‐ribofuranoside (AICAR), A‐769662, and metformin. The anti‐proliferative action of AICAR was concentration‐dependent, reversed by the adenosine kinase inhibitor 5’‐iodotubercidin, but unaffected by the nitric oxide synthase inhibitor, methyl‐L‐arginine. Infection of ECs with an adenovirus expressing constitutively active AMPKα also blocked EC proliferation. In addition, AMPK activation resulted in a marked decline in DNA synthesis that was associated with the arrest of ECs in the G0/G1 phase of the cell cycle. In conclusion, this study demonstrates that AMPK activation inhibits serum‐stimulated EC proliferation by blocking cell cycle progression. The ability of AMPK to inhibit EC growth may provide another mechanism by which this kinase preserves cellular energy during periods of metabolic stress. Supported by NIH grant HL59976.
Reduction of contractions to phenylephrine by L‐NAME in the carotid artery of mice with endothelial overexpression of endothelin‐1
Authors: Baretella Oliver , Li François YL , Chung Sookja K , Vanhoutte Paul M
Published: June 2021

The release of endothelium‐derived contracting factors (EDCF) is increased by exogenous endothelin‐1 (ET‐1). Experiments were designed to determine whether or not endothelial overexpression of ET‐1 affects vascular responsiveness. Carotid artery rings with endothelium of control mice and mice with endothelial overexpression of ET‐1 (TET‐1) were contracted with the α1‐adrenoceptor agonist phenylephrine in the absence or presence of the eNOS inhibitor L‐NAME and exposed to acetylcholine. After washout, the rings were contracted with increasing concentrations of the TP receptor agonist U46619. EDCF‐mediated increases in tension caused by acetylcholine were augmented in TET‐1 arteries compared to controls both in the absence and presence of LNAME. In the presence of the latter, TET‐1 rings showed stronger contractions to U46619 than controls. By contrast, contractions to phenylephrine were reduced by L‐NAME in TET‐1 arteries with a rightward shift and loss of the sigmoidal shape of the concentration‐response curve. These results imply a role of endothelial ET‐1 in EDCF‐mediated contractions and indicate an increased TXA2 receptor signaling in vascular smooth muscle cells. They do not provide an explanation for the decreased response to α1‐adrenergic activation in TET‐1 arteries in the absence of NO. Sponsored by the Swiss National Science Foundation and the Hong Kong Research Grant Council.
Hydrogen peroxide mediates hypoxic induction of arginase II in human microvascular pulmonary endothelial cells
Authors: Chicoine Louis G , Nelin Leif D , Han Zhaosheng
Published: June 2021

Hypoxia induced up‐regulation of arginase II (Arg II) in endothelial cells (ECs) has been recently reported, however, the underlying mechanism(s) is largely unknown. We hypothesized that reactive oxygen species (ROS), in particular H2O2, may be involved. Human lung microvascular ECs were subjected to hypoxia (1% O2, 5% CO2, bal N2) for 24–48h and harvested for immune‐blot analysis, which showed a one‐ and three‐fold increase in ECs Arg II expression, respectively. The experiment was repeated in the presence of each of the following: L‐NAME (1mM), N‐Acetyl‐Cysteine (NAC, 1mM), PEG‐SOD (100 μ/ml), PEG‐Catalase (200 μ/ml) and uric acid (UA, 100μM). We observed that Arg II expression was abolished in the presence of NAC and not altered by L‐NAME indicating that ROS played a role in hypoxia induced Arg II up‐regulation. Treatment with PEG‐SOD demonstrated enhanced Arg II expression, while PEG‐Catalase prevented hypoxic up‐regulation of Arg II. UA did not attenuate hypoxia induced up‐regulation of Arg II indicating that H2O2, but not superoxide (O2−) nor peroxynitrite (ONOO−), played a role in hypoxia induced Arg II up‐regulation. ECs treated directly with H2O2 (1 and 50 μm) for 48h showed that Arg II expression was increased in a dose‐dependent manner. We investigated the cellular source of H2O2 utilizing NADPH oxidase inhibition with diphenylene iodonium, 50μM, as well as with mitochondrial complex inhibition with rotenone 2μM and with antimycin A 10μM. These inhibitors had no effect on hypoxia‐induced Arg II over‐expression. Finally, xanthine oxidase inhibition with oxypurinol, 20μM, showed marked decreases in hypoxia‐induced Arg II over expression. Taken together, these data support our conclusion that xanthine oxidase derived H2O2 mediate, at least in part, Arg II up‐regulation in human lung microvascular ECs exposed to hypoxia.
Mitochondrial DNA Damage‐associated Molecular Patterns are Released in Response to Oxidant Stress in Rat Pulmonary Artery Endothelial Cells
Authors: Hill Jamie K. , Mulekar Sujata , Pastukh Viktor M. , Gillespie Mark N.
Published: June 2021

Sequences of mitochondrial DNA called damage‐associated molecular patterns (mtDNA DAMPs) are released as a consequence of severe trauma into the circulation, where they prime neutrophils and activate target cells in distant organs thereby leading to multi‐organ system failure. The stimulus for mtDNA DAMP formation is unknown. Using cultured rat pulmonary artery endothelial cells, we tested the hypothesis that oxidative mtDNA damage triggers mtDNA DAMP release. Cells were challenged with a non‐cytotoxic concentration of glucose oxidase (GOX) for 15 or 60 minutes. Both qRT‐PCR assessment and a neutrophil‐based bioassay revealed that mtDNA increased in culture medium at 15 min after GOX challenge, but returned to baseline levels within 60 minutes. Interestingly, released mtDNA was detected as fragments rather than intact genome. Culture medium content of the 28S rRNA nuclear gene sequence was unaffected by oxidant challenge. To determine if mtDNA damage was required for mtDNA DAMP accumulation, an adenoviral construct encoding a mitochondria‐targeted DNA glycosylase Ogg1 was used to enhance repair of oxidative mtDNA damage. In these cells, oxidant stress did not increase abundance of mtDNA sequences in culture medium. These observations suggest that oxidative damage to the mitochondrial genome may serve as a stimulus for formation and release of neutrophil‐activating mtDNA DAMPs. Supported by NIH.
BH4 Improves Postprandial FMD in Older Adults
Authors: McCarthy Cameron , Bass Leon , Greer Steven , Harris Ryan
Published: June 2021

Menopause is associated with an increased risk for cardiovascular disease (CVD) and is likely related to a decrease in nitric oxide (NO) bioavailability. The flow mediated dilation (FMD) test is a non‐invasive assessment of NO bioavailability in humans. Tetrahydrobiopterin (BH4) represents a therapeutic agent that may offer a novel means to increase NO bioavailability, preserve vascular integrity, and reduce overall CVD risk. Accordingly, we hypothesized that the ingestion of BH4 will counteract the attenuation in NO bioavailability typically observed following a high fat meal (HFM). In a randomized, double‐blinded, crossover design, 22 older adults (9 postmenopausal women within 3 years, 6 postmenopausal women for greater than 10 years, and 7 men) ingested a HFM with and without the co‐ingestion of BH4 (5 mg·kg−1 of body weight dissolved in apple juice). FMD was measured at baseline and 4 hours post‐ingestion in each trial. A significant treatment effect for delta (Δ) FMD was identified (p=0.048). Specifically, an increase in FMD was observed following the HFM with BH4 (5.4±0.8% to 6.5±1.0%; Δ1.1±0.7%), whereas a slight decrease in FMD was observed following the HFM alone (6.7±0.7% to 6.0±0.7%; Δ−0.7±0.6%). In summary, co‐ingestion of BH4 with a HFM improves postprandial FMD, regardless of postmenopausal status or sex.
The role of inflammation and adaptive immunity in aortic stiffening
Authors: WU JING , Thabet Salim , Chen Wei , Goldstein Anna , Madhur Meena , Hudson Billy , Harrison David
Published: June 2021

Clinical studies show an association between inflammation and arterial stiffness. We have previously demonstrated that T lymphocytes mediate experimental hypertension and vascular dysfunction in various models. In addition, T cells‐derived pro‐inflammatory cytokines such as IL‐17 and TGF‐β are known to induce fibrosis. We therefore propose that hypertensive stimuli induce T cell activation and the secretion of cytokines promote aortic stiffening in hypertension. Using masson trichrome staining, we initially observed profound collagen deposition in the aortic adventitia in C57Bl/6J mice receiving chronic angiotensin II infusion (490ng/kg/min, 14 days). We then biochemically determined the alteration of ECM proteins using hydroxyproline assay and ninhydrin assay. Angiotensin II treatment significantly increased collagen content in the aortas of C57Bl/6J mice by more than two fold (1.3±0.1 μg/mm v.s. 3.5 ±0.3 μg/mm, p<0.01) and this increase was further amplified in Recombination Activating Gene‐1 knockout (Rag1−/−) mice lacking mature lymphocytes (1.3±0.2 μg/mm v.s. 5.6±0.8 μg/mm, p<0.01). Although elastin content was not reduced, the elastin to collagen ratio was significantly depressed by angiotensin II infusion in both strains (23.4±1.2 v.s. 9.5±0.9, p<0.01; 25.5±3.5 v.s. 7.5±0.9, p<0.01). Consistently, angiotensin II increased aortic wall thickness (p<0.01), media to lumen ratio (p<0.01) while decreased extensibility index (p<0.01) in C57Bl/6J mice. These changes in pathology and mechanical properties were less impressive in Rag1−/− mice (p<0.01 for all three parameters). Collectively, our data demonstrate that arterial stiffening might be caused by alteration in ECM proteins, vascular hypertrophy and loss of elasticity. There is clearly an involvement of inflammation and adaptive immunity in the pathogenesis of aortic stiffening.
B(a)P exposure in utero result in oxidative stress‐induced high blood pressure in LEH rat offspring
Authors: Jules George E , Pratap Siddharth , Ramesh Aramandla , Hood Darryl B
Published: June 2021

Cancer Biology, Meharry Medical College, Nashville, TN In utero exposure of fetuses to benzo(a)pyrene [B(a)P], a polycyclic aromatic hydrocarbon, can result in the impairment of brain development and function. Although other developmental indices are thought to be affected, the true nature of the effect of B(a)P exposure in utero is relatively unclear. To investigate the effects of in utero B(a)P exposure on high blood pressure development in later life, timed‐pregnant Long Evans Hooded (LEH) rats were exposed to B(a)P (0, 600 and 1200 μg/kg BW) by oral gavage on embryonic (E) days 14–17. The results indicate that the blood pressure of the B(a)P exposed offspring was significantly elevated compared to the controls. Similarly, western blot analysis and quantitative real time PCR (qRT‐PCR) also showed an increase in the level of protein and mRNA expression for angiotensin (AGT), (6R)‐5,6,7,8‐tetrahydro‐L‐biopterin (BH4) and nitric oxide synthase (NOS). However, there were no significant effects of in utero B(a)P exposure on the number of pups born per litter, the initial and final heart to body weight ratios, or the pre‐weaning growth weights. These data suggest that in utero exposure to B(a)P predispose offspring to functional deficits in cardiovascular development which leads to cardiovascular dysfunction in later life.
Oxidative stress contributes to the cigarette smoke extract induced adverse functional effects of rat cardiac stem cells
Authors: Sumanasekera Wasana K. , Tran David , Rokosh Gregg , Sumanasekera Thimira , Tran Hieu T.
Published: June 2021

Cigarette smoke (CS) is a causative agent in several cardiovascular diseases and premature death. CS is composed of many constituents that can modulate cardiac cell functions. The integrity of cardiac cell functions is crucial in maintaining proper cardiovascular health. The main objective of our study is to investigate whether cigarette smoke extracts (CSE) cause impairment of cardiac stem cell functions via oxidative damage. We hypothesized that CSE, via oxidative modifications of proteins and generation of reactive oxygen species can modulate rat cardiac stem cell (RCSC) functions. This is a novel idea and there are few reports discussing CSE induced impairment of endothelial progenitor cell functions via oxidative stress, but not in the context of cardiac stem cells. In our study c‐kit positive RCSC were treated with cigarette smoke extracts (CSE) for 24 hours, and modulations of RCSC functions were assayed. CSE attenuated RCSC viability, migration, and accelerated apoptosis in a dose dependent manner. Anti‐oxidant vitamin C has prevented CSE induced effects. To find out whether oxidative stress play a role in CSE mediated adverse effects, oxidative modification of proteins due to CSE was detected using Oxiblot ™ assay. Compared to control, in CSE treated cells carbonyl groups of proteins were oxidatively modified. The possibility of vitamin C mediated prevention of CSE induced oxidative modification of proteins is currently under investigation. CSE induced generation of reactive oxygen species will be detected using cytochrome C superoxide assay. Research support was provided by Sullivan University College of Pharmacy faculty development grant awarded to Dr. Wasana Sumanasekera.