bluuebunny/crossref_metadata_2025 huggingface dataset:
Cardiac restricted overexpression of E2F6 alters cell cycle dynamics and results in dilated cardiomyopathy in mice
Authors: Westendorp Bart , Nader Moni , Salih Maysoon , Leenen Frans H , Tuana Balwant S
Published: June 2021

The E2F transcription factors play a critical role in cell cycle control and developing myocardium. Cardiomyocytes undergo permanent cell cycle arrest shortly after birth and we investigated the role of E2F6, a repressor of E2F‐responsive genes, by generating transgenic (Tg) mice with cardiac‐specific expression of E2F6 driven by the alpha‐myosin heavy chain promoter. Three independent Tg lines with different levels of E2F6 (~3‐, 6‐ and 9‐fold vs wild type) were established, which showed mortality and CHF symptoms in young adults, with increasing severity correlating with E2F6 levels. Echocardiography showed progressive LV dilation and decreasing ejection fractions in Tg mice. E2F6 targeted the nucleus with a profound reduction in phospho‐histone H3 staining but no changes in apoptosis in 4‐day‐old Tg hearts. Increases in the G1/S phase markers cyclin D3, cdk4, p‐Rb were noted while the G2/M marker p‐cdc2 did not change in Tg hearts. Heterochromatin formation was attenuated in Tg hearts, as reflected by reduced methylated histone H3, and histone deacetylase 4 protein. These data suggest that regulated expression of E2F6 is critical for normal cardiac development and function, since overexpression alters chromatin remodeling and cell cycle dynamics, resulting in dilated cardiomyopathy. (Supported by CIHR and HSFO).
Regenerative medicine based on muscle stem cells
Authors: Huard Johnny
Published: June 2021

Members of our laboratory have isolated various populations of myogenic cells from the postnatal skeletal muscle of normal mice by examining the cells’ adhesion characteristics, proliferation behavior, and myogenic and stem cell marker expression profiles. Although most of these cell populations have displayed characteristics similar to those of satellite cells, we also have identified a unique population of muscle‐derived stem cells (MDSCs). MDSCs exhibit long‐term proliferation and high self‐renewal rates, high resistance to stress, and can differentiate toward various lineages, including muscle (skeletal and cardiac), neural, endothelial, osteogenic, and chondrogenic lineages, both in vitro and in vivo. The transplantation of MDSCs, in contrast to that of other myogenic cells, has improved the efficiency of dystrophic muscle regeneration and the delivery of dystrophin to dystrophic muscle. I will also show that transplantation of female MDSCs (F‐MDSC) rather than male MDSCs (M‐MDSCs) significantly improves skeletal muscle regeneration despite a similar myogenic and stem cells markers expression by both cell types. My presentation will also address the influence of environmental cues released within dystrophic or injured skeletal muscle upon the differentiation of MDSCs toward fibrotic cells. I will discuss potential strategies by which scar tissue formation can be prevented within injured muscle through TGF‐β1 blockade. I then will discuss the use of MDSCs in gene therapy and tissue engineering applications designed to improve bone and cartilage healing through the genetic modification of MDSCs to express osteogenic proteins (BMP‐2 and ‐4) as well as angiogenic factor (VEGF). The results outlined in my presentation open new avenues by which researchers could use muscle stem cell–based gene therapy and tissue engineering to improve tissue regeneration.
Neuro‐vascular interaction during mouse development
Authors: Mukoyama Yosuke
Published: June 2021

Nerves and blood vessels provide a vital network among tissues. We have demonstrated that arteries align themselves with developing peripheral nerves in the embryonic limb skin. Moreover, recent experiments using the Cre‐loxP recombination system to specifically inactivate Vegf‐A in peripheral nerves or Np1, a co‐receptor for VEGF‐A, in endothelial cells suggest that nerve‐derived VEGF‐A promotes arterial differentiation via NP1‐mediated positive‐feedback loop. Nerve‐blood vessel alignment is unperturbed in these mutants, suggesting the involvement of distinct nerve‐derived signals in the patterning and arterial differentiation process. Indeed, we have begun to test the functional role of nerve‐derived chemokine in the nerve‐arterial vessel alignment. These observations will allow us to further dissect the process of angiogenesis in the limb skin. Our studies have also extended to elucidating the neuro‐vascular interaction in developing heart and spinal cord. In the heart, sympathetic neurons innervate and control coronary blood vessels as well as cardiac muscle, whereas sprouting angiogenesis develops early vascular patterns in the spinal cord. These preliminary data will be discussed.
Single‐Molecule FRET Observations of Allosteric Regulation of Ribosome Function
Authors: Blanchard Scott C , Munro James B
Published: June 2021

High‐spatial and ‐time resolution single‐molecule fluorescence resonance energy transfer measurements have been used to probe the structural and kinetic parameters of transfer RNA (tRNA) movements within the ribosome critical to direction translocation along messenger RNA. Direct observations of tRNA and ribosome domain motions, quantified on wild‐type and mutant ribosome complexes, reveal that the ribosome is in dynamic exchange between multiple metastable configurations that depend strongly on the nature of bound substrates. This new dynamic information suggests a framework in which the formation of intermediate states in the translocation process is achieved through global conformational rearrangements intrinsic to the ribosome particle.
Sphingosine Kinase 2, an Evil Kinase?
Authors: Spiegel Sarah , Hait Nitai C.
Published: June 2021

Sphingosine 1‐phosphate (S1P) is a potent lipid mediator that regulates a variety of biological responses important for cancer progression. Two sphingosine kinase isozymes SphK1 and SphK2 control S1P levels. While SphK1 promotes growth and survival and has been linked to cancer progression, much less is known about the functions of SphK2. EGF, an important growth factor required for breast cancer progression, activates both SphK1 and SphK2 in MDA‐MB 453 human breast cancer cells. Moreover, expression of both SphK1 and SphK2 are required for EGF‐directed motility. EGF induced rapid phosphorylation of SphK2 which required ERK1. ERK1 phosphorylated SphK2, increased its enzymatic activity, and was associated with SphK2 in cells. Site‐directed mutagenesis indicated that SphK2 is phosphorylated on Ser351 and Thr578 by ERK1 and that phosphorylation of these residues is important for EGF‐stimulated migration of MDA‐MB‐453 cells. Expression of SphK2, which is enriched in the nucleus of MCF7 human breast cancer cells, increased cyclin‐dependent kinase inhibitor p21 but had no effect on p53 or its phosphorylation. Downregulation of endogenous SphK2 decreased the anti‐cancer drug doxorubicin‐induced expression of p21 without affecting increased p53. It also decreased G2/M arrest and markedly enhanced apoptosis induced by doxorubicin. siSphK2 also reduced doxorubicin‐induced p21 expression in p53‐inactivated MCF‐7 cells. These results demonstrate that endogenous SphK2 is important for p53‐independent induction of p21 expression by doxorubicin and suggest that it may regulate the balance between cytostasis and apoptosis of human cancer cells. Supported by 37GM043880 and R01CA61774.
Effect of a culturally and language‐sensitive diabetes education on dietary changes and plasma lipid profile in Hispanic diabetics
Authors: Ojo Emmanuel , Beeson Lawrence , DeLeon Marino , Cordero‐MacIntyre Zaida
Published: June 2021

O: To assess effects of diabetes education on dietary changes and plasma lipid profile.

M: Hispanic subjects(n=13 F and 13 M, mean age=53.8±12.7) participated in a 3‐month study. Spearman correlation coefficient was used to evaluate correlations between dietary intake and laboratory measurements.

R: At baseline, % calories, protein(r=0.408, p=0.038) was correlated with plasma HDL(mg/dL). At 3 months, plasma triglycerides(mg/dL) correlated with protein(g)(r=−0.43, p=0.03), % calories, protein(r=−0.43, p=0.028), and % calories, carbohydrate(r=0.41, p=0.039); plasma HDL was correlated with % calories, total fat(r=0.56, p=0.003), % calories, saturated fat(r=0.48, p=0.012), % calories, monounsaturated fatty acids(r=0.587, p=0.002), supplementary chromium(μg)(r=0.56, p=0.003), % calories, protein(r=−0.57, p=0.002), % calories, carbohydrate(r=−0.67, p=0.0002). A decrease in alcohol(g)(r=−0.46, p=0.02), % calories, alcohol(r=−0.43, p=0.03) and increase in % calories, protein(r=0.39, p=0.046), was associated with a significant increase in plasma HDL. Decrease in alcohol (r=0.44, p=0.025), % calories, carbohydrate (r=0.45, p=0.02), and % calories, alcohol (r=0.45, p=0.02) was associated with a significant decrease in plasma triglycerides.

C: Dietary changes were significantly associated with some lipid profile parameters after a 3‐month diabetes education.
Weight and lean mass as the major predictors of resting energy expenditure in Mexican Adults
Authors: Anaya‐Loyola Miriam Aracely , Guerrero Adriana Lopez , Martinez Ma. Guadalupe
Published: June 2021

Resting energy expenditure (REE) is important to establish the energy requirements and adequate energy balance and to predict overweight and obesity as risk factors of chronic diseases. In this study the relationship between body composition and REE was evaluated, in a total of 208 adults living in the urban area of Querétaro. Anthropometric data include weight, height, circumference of waist and hips and body mass index (BMI). Body composition was evaluated by mean of DEXA and REE by indirect calorimetry. Average age, weight and height were 51.9 ± 8.3 y, 71.1±12.0 kg and 157.2±8.8 cm, respectively. Mean REE was 1421.0 ± 284.7 kcal/d and mean VO2 was 2.9±0.4 mL/kg/min. Mean body fat was 36.6 % (higher in women), which was distributed mainly in trunk and lower extremities. REE was strongly correlated to lean mass in trunk (r=0.758, P=0.0001), waist (r=0.56, P<0.0001) and BMI (r=0.39, P=0.0001). Prevalence for overweight and obesity were 44.3% and 37.5%. Age did not affect REE, neither BMI nor percentage of body fat. The percentage of body fat was higher in subjects with hypertension and those presenting at the same time hypertension and diabetes. Multiple regression analysis showed that weight and lean mass were the main predictors of REE.
The effect of increased dietary calcium on fecal fat excretion in overweight and obese adolescents
Authors: Martin Berdine R , Singh Rajni , Campbell Wayne W , Teegarden Dorothy , Weaver Connie
Published: June 2021

This study was designed to measure the effect of calcium supplementation from a dairy vs. calcium carbonate source on fecal fat excretion in overweight and obese adolescent boys and girls. Twenty girls (BMI 34+/−5 kg/m2) and fourteen boys (BMI 28 +/−5 kg/m2), aged 12–15 y, participated in two 3 week controlled feeding trials separated by a 3‐week washout period. Total dietary intakes during both sessions were eucaloric and macronutrient matched for energy needs. Intake included a basal diet containing 759 mg Ca/d plus frozen products based on soy protein with no added Ca (control) or the basal diet plus frozen products based on either dairy or soy protein containing added Ca (714 mg/d) in a cross‐over design. All urine and feces were collected during each session. Twenty‐four h diet and fecal samples were analyzed for Ca by inductively coupled plasma spectrophotometry. Crude fat of dried diet and feces was extracted using an automated Soxhlet type system. Fractional fecal fat was not influenced by increased Ca intake from dairy or CaCO3. However, pooled data indicated that fractional fecal fat excretion was positively related to fecal Ca excretion.

Supported by NIH DK066108, National Dairy Council and test products were a gift from The Schwan Food Company.
Gait and balance disturbances in older adults with sarcopenic‐obesity
Authors: Waters Debra Lynn , Grant Andrea , Herbison Peter , Hale Leigh , Goulding Ailsa
Published: June 2021

Low lean body mass (sarcopenia,SS) and high fat mass (obesity,OO) are independent factors associated with gait and balance disturbances during aging. The evidence for sarcopenic‐obesity (low lean with high fat mass) is less clear and the focus of this investigation. Subjects (49 M, 134 F, 73 ± 6yr) were recruited from a falls prevention intervention trial at baseline when timed up and go (TUG), 30s chair stand (CS), and step test (ST) were assessed. Appendicular skeletal muscle mass (ASM), fat mass, and neck of femur bone mineral density were measured by DXA (Lunar DPX‐L) scanner. Body composition phenotypes were classified from lean and fat mass (normal lean, NL). The CS was different between the groups (p=0.03) and due to lower scores in SO compared to NL and OO (p=0.005, p=0.001, respectively). ST was significantly different (p=0.03) and due to lower scores in the SO compared to NL, OO, and SS (p=0.03, p=0.008, p=0.014, respectively). TUG was slower in SO compared to NL and OO (p=0.005, p=0.02, respectively). Neck of the femur t‐score in SO males was poorer than SO females (p=0.0009) and femoral neck bone mineral density and low ASM were independently associated with lower CS score (p=0.005, p<0.0001, respectively) with a trend towards an interaction with fat mass (p=0.06). This data provide evidence that sarcopenic‐obesity is associated with gait and balance deficits and possibly has a relationship with poor bone health. Supported by Accident Compensation Corporation New Zealand and University of Otago Research Grants.
Carbohydrate restriction decreases small LDL particles, attenuates inflammation and alters mechanical properties of aortas in the cholesterol fed guinea pig
Authors: Torres‐Gonzalez Moises , Leite Jose O , Volek Jeff S , Fraser Heather , Fernandez Maria Luz
Published: June 2021

Both dietary cholesterol (chol) and carbohydrate restriction (CR) significantly impact plasma lipids, atherogenicity of lipoproteins and inflammatory cytokines. We evaluated the effects of chol and CR on aortic cholesterol and triglycerides accumulation, plasma secretory phospholipase A2 (sPLA2) activity, aorta stiffness as well as aortic cytokine concentration in guinea pigs. Forty adult male guinea pigs (10/group) were fed either low (L) (0.04 g/100g) or high (H) (0.25g/100g) amounts of chol in combination with CR (10% total energy) or high carbohydrates (54% total energy) for 12 wks. Groups fed the high chol (control‐H and CR‐H) had higher cholesterol and triglycerides accumulated in aortas (P< 0.05) and higher sPLA2 activity than those guinea pigs in the L chol groups. CR resulted in lower concentrations of plasma small LDL subfractions (P < 0.01) and aortic cytokines and chemokynes including TNF‐α, IL6 and MPC‐1 (P < 0.01) than the high carbohydrate groups. Regarding the aorta stiffness, the control‐H and the CR‐L were stiffer than the control‐L and the CR‐H groups. These results can be interpreted as different stages of atherosclerosis development. The control‐H diet is postulated to be in the last stage of atherosclerosis, the CR‐H and the control‐L in an intermediate phase and the CR‐L at the beginning of the process. These results demonstrate that CR has a major impact on atherogenicity of lipoproteins, aorta stiffness and aortic cytokine accumulation. In addition, CR diets appeared to ameliorate the atherosclerotic potential of the high cholesterol diets.
Short‐term Changes in Lipoprotein Subclasses and C‐reactive Protein Levels with the Low Carbohydrate and Low‐Fat Diets
Authors: Tangney Christy C , Stoernell Colene Renee , Rockway Susie W
Published: June 2021

Background: Diets designed to promote weight loss and improve atherogenic lipid profiles traditionally include a reduction in total fat and in particular, saturated fats. This study was designed to test the efficacy of a low‐fat diet vs. a carbohydrate restricted (low‐CHO) diet in hypertriglyceridemic patients on lipid profile, weight loss, C‐reactive protein (CRP) and satiety.

Methods: Twenty‐eight hypertriglyceridemic subjects (with fasting triglyceride (TG) levels exceeding 1.69 mmol/L) were randomized to either the low‐CHO or low‐fat diet for 8 weeks. Fasting bloods were drawn at weeks 0 and 8 and analyzed for lipids via NMR and CRP. Anthropometric measurements were also obtained. Three random 24‐hr food recalls were used to assess compliance during the trial.

Results: Based on the 23 subjects who completed the protocol, small LDL particles of the low‐CHO subjects fell by 46% after 8 weeks, while those on the low‐fat plan exhibited an increase (36.5%), p=0.045. An average decrease in TG of 18% among low‐CHO subjects in contrast to an increase of 4% for low‐fat group was not significantly different. Nor were there significant differences in CRP, overall dietary compliance, satiety or the magnitude of body weight lost between groups (low‐CHO group, −3.8% vs low‐Fat group, −1.6%).

Conclusion: Favorable reductions in small LDL concentrations after 8 weeks suggest that a moderately restricted carbohydrate diet (less than 20% CHO as energy) can promote a healthier, less atherogenic lipid profile when compared to the low‐fat diet.
Responses of blood lipids to consumption of honey versus sucrose in adult men and women
Authors: Altamimi Alia , Petrisko Yumi , Hong Mee Young , Rezende Lauren , Kern Mark
Published: June 2021

Consumption of added sugars in the USA is about twice the recommended intake and has been linked to chronic diseases such as atherosclerosis. Recent research has suggested that relative to sugar, honey does not produce similar adverse responses; however, the trials were of short duration and samples sizes were small. We assessed the short‐term (1 week) and relatively long‐term (1 month) effects of clover honey compared to sucrose on changes in lipid concentrations. Thirty‐seven subjects (21 men and 16 women) aged 24–55 years (BMI=17.6–35.8 kg/m2) participated in two 4‐week random trials in a balanced, cross‐over design separated by at least a 4‐week washout period. During each trial, subjects consumed either clover honey or sucrose at a rate of 1.2 g/kg/day under free‐living conditions with instructions to avoid changing their habitual food intake. Serum triglyceride (TG) concentrations were elevated (p ≤ 0.05) after one week for both trials but remained elevated (p ≤ 0.05) at the 4‐week timepoint during sucrose consumption only. The elevation after 1 week during the honey trial was concurrent with a transient increase (p ≤ 0.05) in body weight. No effects (p > 0.05) on serum concentrations of total cholesterol, LDL‐C, or HDL‐C were detected for either trial. These data suggest that honey may serve as a wise substitute for sugar with regard to adverse effects on serum triglyceride concentrations.
Cortisol Changes with Weight Loss and Affects Food Intake from a Buffet
Authors: Witbracht Megan Gage , Olney Deanna K. , Horn William F. , Adams Sean H. , Keim Nancy L. , Laugero Kevin , Van Loan Marta
Published: June 2021

The purpose of this study was to determine if weight (wt) loss alters cortisol (COR) rhythm and if an association exists between [COR] and self‐selected food intake following an energy‐restricted diet. Obese men (n=2) and women (n=14) participated in this 15‐wk trial. All foods/beverages were provided and measured to achieve wt maintenance during 3‐wk baseline and wt loss during 12‐wk energy‐restriction (500 kcal/d reduced). Wt, body fat, intra‐abdominal fat (IAAT) were measured, and subjects participated in a test protocol that included ingestion of standardized breakfast and lunch and an ad libitum buffet‐style dinner at wks 3 & 15. Saliva was collected 10 times from waking to bedtime on test days. At wk 15 significant changes in wt, fat mass and IAAT occurred and averaged −6.4 ± 0.8 kg, −5.2 ± 0.6 kg, and −7.6 ± 1.6 cc. Peak salivary [COR] was reduced significantly, from 17.12 ± 1.99 at wk 3 to 12.97 ± 1.10 nmol/L at wk 15. Times of peak and nadir COR did not differ at wks 3 & 15, suggesting the absence of phase shift in COR rhythm. Buffet food intake was variable (kcal range=572 to 2636). Salivary [COR] measured immediately before and 30 min after dinner were positively associated with kcal intake (r=.50; p<0.05) and (r=0.72; p<0.01). Energy restriction and/or wt loss is associated with change in amplitude of COR rhythm. Also, [COR] around the meal and kcal consumed at the meal influence one another. Funded by Dairy Mgmt Inc & USDA.
Correction of dyslipidemia resulting from high fat diets with purified anthocyanins from blueberry or strawberry but not in context of the complete berry
Authors: Prior Ronald L. , Wilkes Samuel , Wu Xianli
Published: June 2021

Male C57BL/6 mice received diets with either 10% kcal from fat (LF), a high fat diet [45% (HF45) or 60% (HF60) kcal from fat]. In the first study the diets were prepared with or without freeze dried powders from whole blueberries (BB) or strawberries (SB). In the 2nd study, a LF or HF60 diet was fed and purified anthocyanins (ACNs) from SB or BB (1 mg total ACN/mL) were added to the drinking water of the treatment groups fed HF60 diet. In Study 1, when whole SB or BB powder was included in the diet, plasma triglycerides were increased by feeding the HF diet but were elevated further when BB was included in the HF diet. Liver total lipids and triglycerides were increased in mice fed HF diet but were not altered by either SB or BB in the diet. Liver cholesterol was not altered by dietary fat level or SB or BB in the diet. In the 2nd study, after 8 weeks mice fed the HF60 diet plus purified ACNs from BB or SB in the water, had lower body weight gains and body fat than the HF60 fed controls. Remarkably, plasma cholesterol (mg/dL) was 128±12, 238±20, 151±18, and 166±10 and triglycerides (mg/dL) were 173±23, 312±39, 168±16 and 154±33 in LF Control, HF60, HF60‐BB and HF60‐SB, respectively. Plasma cholesterol and triglyceride levels were elevated with a high fat diet and decreased to control levels when purified ACNs from either SB or BB were included in the drinking water. Purified ACNs prevented the development of obesity, but not in the context of the whole berry.
Effects of n‐3 PUFA supplementation with or without low‐calorie cranberry juice cocktail on flow‐mediated vasodilation in men
Authors: Couillard Charles , Riopel‐Meunier Julie , Lamarche Benoît , Couture Patrick
Published: June 2021

Consumption of fish as well as fruits and vegetables have been shown to have favorable effects on CVD risk. The present study was therefore undertaken in order to compare the effects of fish oil (FO) w/wo cranberry juice cocktail (CJC) supplementation on flow‐mediated vasodilation (FMD) in men. A group of 53 men was divided into 4 groups: 1) placebo, n=11, 2) 500 mL/day CJC, n=13, 3) 2.4 g/day of FO, n=15 and 4) CJC+FO, n=14. Before and after the intervention, FMD was measured by ultrasound echography of the brachial artery at 60 and 90 seconds after hyperemia. We found no difference in baseline brachial artery diameter (AD), FMD60 and FMD90 between the four groups. We noted that FMD60 (1.2 ± 1.8 %, p=0.029) and FMD90 (+2.5 ± 3.2 %, p=0.013) were significantly improved following the intervention in CJC+FO individuals only. CJC subjects showed no improvement in FMD but displayed a significantly increased resting brachial AD (+0.20 ± 0.24 mm, p=0.013) after the intervention. We also found that changes in FMD60 and FMD90 were negatively associated with brachial AD. In summary, we found that CJC+FO supplementation improved FMD in healthy overweight subjects and that drinking CJC on daily basis for 12 weeks was associated with an increase in resting brachial AD. Further studies are needed to confirm our results and verify the clinical relevance of our observations.

Supported by the Canadian Institutes of Health Research (MOP‐64438)
Atherosclerotic intralesional haemorrhage moulds monocyte differentiation into a macrophage antioxidant phenotype bearing the hemoglobin scavenger receptor CD163
Authors: Boyle Joseph J , Philippidis Pandelis , Horncastle Donna , Cantilena Sandra , Leung Viola , Taylor Kenneth M , Landis Clive , Haskard Dorian O
Published: June 2021

Atherosclerotic intralesional haemorrhage promotes plaque progression. We investigated macrophage differentiation in atherosclerotic plaques with intralesional hemorrhage and in vitro with hemoglobin:haptoglobin (Hb:Hp) complexes. Macrophages within culprit lesions partitioned into reciprocal populations, with 26.1±2.3% expressing high levels of CD163 but depressed HLA‐DR (CD163high‐HLA‐DRlow phenotype), and the remainder 72.5±2.4% expressing the inverse (classical inflammatory) phenotype (CD163low‐HLA‐DRhigh). CD163high macrophages were associated with areas of hemorrhage (P<0.008), expressed increased (antioxidant) heme oxygenase‐1 (P<0.00001), and were protected from oxidant stress (P<0.003), despite greater iron content (P<0.001). This phenotype was reproduced in vitro by differentiating human monocytes with Hb:Hp, which required phagocytosis of Hb:Hp via CD163 and was inhibited by anti‐IL‐10. The divergent commitment of macrophages to CD163highor CD163low phenotypes was predictively modelled using a systems biology approach incorporating an IL‐10 autocrine positive feedback loop. Our results provide evidence for a novel CD163highmacrophage subset within culprit lesions. This appears to be moulded by the local microenvironment to become ‘fit for purpose’ for clearing Hb. Its therapeutic induction may reduce plaque progression.

Funded by the British Heart Foundation
Polycystin‐1 trafficking from Golgi to cilia
Authors: Ward Heather Hilary , Peterson Brandon , Romero Elsa , Deretic Dusanka , Wandinger‐Ness Angela
Published: June 2021

Alterations in location and function of cellular proteins contribute to renal cyst development. Polycystin‐1 (PC‐1) mutations cause autosomal dominant polycystic kidney disease (ADPKD). PC‐1 localizes to primary cilia of renal epithelial cells, where it participates in signaling events that instruct cells to maintain a mature state within tubules. Though ciliary PC‐1 location and function is lost in ADPKD, the mechanisms that direct PC‐1 to cilia are unknown. We identified and tested the function of a VxPx sequence at the C‐terminus of PC‐1, which is analogous to the VxPx motif in polycystin‐2 and rhodopsin. The PC‐1 VxPx motif is a recognition site for a multi‐protein complex containing ARF4, development and differentiation enhancing factor 1 (DDEF1), Rab6, Rab8, and Rab11 that mediates trafficking of PC‐1 from Golgi to renal epithelial primary cilia. PC‐1, DDEF1, and Rab11 co‐localize in normal human renal epithelial cells on Golgi subdomains that may represent exit sites. Immunoprecipitation studies show Golgi localized PC‐1 exists in a complex with DDEF1 and ARF4‐GFP, while PC‐1 at the plasma membrane complexed to E‐cadherin does not associate with DDEF1. Interaction is dependent on the integrity of the VxPx motif. We conclude that specialized targeting machinery is recruited by the VxPx motif and is critical for trafficking PC‐1 from the Golgi to primary cilia. This research is supported by NIH grant DK50141.
Loss of CXC chemokine receptor 3 signaling cause a delay in epidermal and dermal maturation and leads to hypertrophic scarring
Authors: Yates Cecelia Christina , Whaley Diana , Bodnar Richard , Wells Alan
Published: June 2021

CXCR3 and its ligands modulate the timing of keratinocytes migration, fibroblasts and endothelial cell immigration into the wound and epidermal and matrix maturation during wound healing. To investigated the long term effects of CXCR3 in wound healing full thickness excisional wounds were created on CXCR3−/− or wildtype mice and examined at 180 days. Grossly the CXCR3−/− mice presented a thick keratinized eschar compared to the wildtype mice that had a scarcely noticeable scar. Histological analysis of the CXCR3−/− mice also revealed a thicker epidermis that was hyperproliferative, dermal layers that were disorganized with thick and long collagen fibrils, and contained excessive collagen content in comparison to the wildtype mice. Interestingly, the CXCR3−/− wounds results in a weaker tensile strength as a result of decreased cross‐linking of collagen fibers. A lack of ECM turnover was shown by the elevated expression of matrix proteins tenascin C, fibronectin, and an imbalance of MMP‐9 expression; these were noted as late as 180 days postwounding, whereas these markers of an immature dermis are long gone in wounds in wildtype mice. These in vivo studies establish that the absence of the CXCR3 signaling network results in hypertrophic scarring by delaying maturation and altering the synthesis and degradation of collagen in addition to other matrix components during wound healing. Supported by grants from the NIH
Coronin 1C negatively regulates cell migration in intestinal epithelial cells
Authors: Samarin Stanislav Nikolayevich , Nusrat Asma
Published: June 2021

The coronin family of actin‐binding proteins consisting of seven proteins in mammals is known to play a critical role in various actin‐driven processes. Intestinal epithelial cells (IECs) abundantly express a member of coronin protein family, Coronin 1C. Since actin cytoskeletal restructuring is required for cell migration observed during pathologic processes, such as wound closure, we investigated the role of Coronin 1C in the regulation of IEC cell migration. Migration of a model intestinal epithelial cell line SK‐CO15 on different extracellular matrixes and cell invasion into a 3 dimensional (3D) matrix was determined. siRNA‐mediated knock‐down of Coronin 1C enhanced the formation of membrane protrusions in cells grown in 3D culture and increased their invasiveness into 3D matrix. Furthermore, the depletion of Coronin 1C significantly increased the rate of actin‐based motility in wounded SK‐CO15 cells by increasing the persistence of membrane protrusion at the leading edge of migrating cells. These findings support a negative regulatory role for Coronin 1C in IEC migration.

This research is supported by the Crohn's and Colitis Foundation of America (for SNS) and NIH (DK55679 for AN).
Integrin‐linked kinase KO mice display abnormal liver histology and hepatomegaly following partial hepatectomy
Authors: Apte Udayan , Gkretsi Vasiliki , Yang Yu , Mars Wendy , Bowen William C. , Dedhar Shoukat , Kaestner Klaus , Michalopoulos George k.
Published: June 2021

Hepatocyte proliferation and differentiation are greatly affected by extracellular matrix (ECM). Integrin‐linked Kinase (ILK) is a cell‐ECM‐adhesions component implicated in cell‐ECM signaling via integrins. We investigated the role of ILK in liver size regulation in vivo by utilizing the LoxP/Cre model system. Targeted deletion of ILK in mouse liver (ILK‐KO) resulted in histological abnormalities in the liver characterized by disorderly hepatic plates, increased hepatocyte proliferation and apoptosis, increased proliferation of biliary cells, and increased deposition of extracellular matrix soon after birth. Microarray analysis indicated upregulation of integrin and matrix‐related genes and a concurrent downregulation of differentiation‐related genes. Western blot analysis indicated change in the expression of many integrins in the liver including integrin β1, β3, β5, and αV. Furthermore, The liver‐targeted ILK‐KO mice had increased liver‐to body‐weight ratio and fail to return to normal liver weight following partial hepatectomy. This increase in liver size was due to prolonged cell proliferation observed in the ILK‐KO mice even at 14 days after partial hepatectomy. Our results highlight the significance of ILK and hepatic ECM‐signaling for hepatocyte proliferation and liver size regulation.
Endogenous CXCL10/Interferon‐γ‐Inducible Protein (IP)‐10 orchestrates myocardial infarct healing
Authors: Bujak Marcin J. , Leucker Thorsten , Zymek Pawel , Veeranna Vikas , Huebener Peter , Tager Andrew , Luster Andrew , Frangogiannis Nikolaos
Published: June 2021

IP‐10, a chemoattractant for effector T cells with angiostatic and anti‐fibrotic properties, is markedly but transiently induced in the infarcted heart. We hypothesized that endogenous IP‐10 may suppress premature angiogenesis and fibrosis in the infarct until the wound is debrided and the local environment can support fibrous tissue deposition. Histomorphometric, molecular and echocardiographic endpoints were compared between IP‐10 −/− and WT mice undergoing reperfused infarction protocols. IP‐10 −/− mice exhibited increased neutrophil and macrophage density in the infarcted area. mRNA expression of the CC chemokine MCP‐1 was significantly higher in IP‐10 −/− infarcts; however, cardiac mRNA levels of the pro‐inflammatory cytokines IL‐1β, TNF‐α and IL‐6 were comparable with WT animals. Myofibroblast density was increased in infarcted IP‐10 −/− hearts resulting in increased collagen deposition in the infarct and the peri‐infarct area. Enhanced inflammation and fibrosis in IP‐10 −/− mice was associated with extension of the scar, worse systolic dysfunction and increased dilation of the infarcted ventricle. IP‐10 deficiency results in disturbed orchestration of post‐infarction cardiac repair, enhanced fibrosis and accentuated adverse remodeling (R01 HL‐76246).
Telmisartan treatment upregulates GTP‐cyclohydrolase I and prevents eNOS uncoupling in streptozotocin‐induced diabetes
Authors: Daiber Andreas , Oelze Matthias , Wenzel Philip , Müller Johanna , Alhamdani Mohamed S.S. , Reifenberg Kurt , Münzel Thomas
Published: June 2021

Cardiovascular complications in patients with diabetes mellitus are associated with increased oxidative stress in vascular tissue. A key event for NO synthase (NOS) uncoupling involves downregulation of BH4 synthesizing enzyme GTP‐cyclohydrolase I (GCH‐I). We here investigated the effects of AT1‐receptor blockade with chronic telmisartan therapy on GCH‐I expression, NOS uncoupling and endothelial function in streptozotocin (STZ)‐induced diabetes mellitus (type I). Diabetes was induced by single i.v. injection of STZ (60mg/kg, 7 weeks) in male Wistar rats (250g). Telmisartan (25mg/kg/d) therapy did not improve blood glucose and body weight. Aorta from diabetic animals had vascular dysfunction as revealed by isometric tension studies. ROS produced by NADPH oxidase, mitochondria and xanthine oxidase were increased in the diabetic group. NADPH oxidase subunits mRNA and protein expression was increased in response to STZ. Importantly, the expression of the GCH‐I and eNOS activating Ser1177 phosphorylation was decreased by STZ. Therapy with telmisartan normalized all these parameters almost completely. The results of the present studies demonstrate for the first time that AT1 receptor treatment of diabetic animals with telmisartan is able to prevent downregulation of the important BH4 synthesizing enzyme GCH‐I, which prevents eNOS uncoupling and an activation of cardiovascular superoxide sources.
Respiratory lethality in newborn NPAS3‐deficient mice
Authors: Zhou Shutang , Degan Simone , Sunday Mary E
Published: June 2021

The neuronal PAS domain protein 3 (NPAS3) is a close homolog of Drosophila Trachealess. By quantitative RT‐PCR, NPAS3 gene expression is highest at E10.5‐E13.5 in developing murine lungs. NPAS3 immunostaining shows positive nuclei in the developing mesenchyme and epithelium. NPAS3 loss‐of‐function embryos (KOs) have deficient lung branching. More than 95% of NPAS3 KO newborns die within 48 hours after birth in respiratory distress with poorly developed alveoli, abnormal distribution of smooth muscle actin along the airways and an increase in intrapulmonary nerve fibers. NPAS3 Heterozygotes (NPAS3‐Hets) have reduced alveolarization at birth as well, which persists in adults. The 4‐month old NPAS3‐Hets show airway hyperreactivity (AHR). Similarly, reduced alveolarization and AHR occur in premature infants with chronic lung disease, called bronchopulmonary dysplasia (BPD). Microarrays and QRT‐PCR demonstrate significantly decreased expression of sonic hedgehog (SHH), Gli2, FGF9, and BMP4 as well as elevated expression of Sprouty2 in NPAS3 KOs. Deficits in any or all of these signaling pathways could contribute to the observed pulmonary lethal phenotype in NPAS3 KOs. Our data suggest that NPAS3 play an important role in lung development and in responses to lung injury.

Supported by NIH Grant HL44984.
Linking of IL‐25 and SCF in allergic airway inflammation
Authors: Dolgachev Vladislav , Berlin Aaron , Lukacs Nicholas W.
Published: June 2021

IL‐25 can directly induce Th2 cytokines, IL‐4, 5 and 13, when injected in vivo in mice and has been linked to allergic responses. Using an established model of chronic allergen‐induced airway disease our studies identified elevated IL‐25 expression in the lung. When dispersed lung cells were activated in vitro with rIL‐25, a significant increase in Th2 cytokines was observed in cells from allergic lungs compared to lung cells from non‐allergic mice with the primary cell that was activated by IL‐25 was c‐kit+ (SCF receptor) cells. Our laboratory has previously demonstrated that neutralization of SCF significantly reduces disease, including AHR, airway remodeling and local Th2 cytokine production. The expression of IL‐25 was inhibited by anti‐SCF in allergic mice suggesting that SCF‐mediated pathways were linked to IL‐25. We found that when rIL‐25 was administered into the airways of allergic mice an exacerbation of disease was induced characterized by increased Th2 cytokines. The treatment of mice with anti‐SCF abrogated the response, further indicating a link of IL‐25‐induced disease with SCF. Furthermore, our studies identified that SCF can directly increase IL‐25 expression in the eosinophils and was dependent upon a PI3K‐induced pathway. Altogether, these studies establish a strong link between the developing allergic response, SCF‐mediated pathways, and IL‐25‐induced responses.
Inhibition of the soluble epoxide hydrolase (sEH) alters vascular remodeling
Authors: Revermann Marc , Barbosa‐Sicard Eduardo , Zwissler Bernhard , Fleming Ingrid , Hammock Bruce , Brandes Ralf Peter
Published: June 2021

Epoxyeicosatrienoic acids (EETs) have potent anti‐inflammatory effects and promote endothelial function. The soluble epoxide hydrolase (sEH) metabolizes EETs to their less active diols (DHETs) and thus inhibition of sEH increases EET concentrations. Since EETs reduce smooth muscle cell (SMC) migration, proliferation and monocyte adhesion to the vascular wall in vitro, we hypothesized that inhibition of sEH using 12‐(3‐adamantan‐1‐yl‐ureido) dodecanoic acid (AUDA) prevents vascular remodeling in hyperlipidemic ApoE−/− mice. Remodeling was studied using models with different inflammatory and SMC‐stimulating properties. AUDA increased the ratio of EETs to DHETs in all models.

The effect of AUDA in the femoral artery cuff model was a significant reduction of inflammation‐accompanied neointima within 2 weeks (43% reduction in intima/media ratio ‐ IMR, p<0,05). Carotid artery ligation induced a mainly SMC‐formed neointima within 4 weeks and this effect was significantly attenuated by AUDA (64% reduction in IMR, p<0,05).

Angiotensin II infusion causes severe aortic inflammation aneurysm formation. In this model AUDA reduced thoracic aorta remodeling (26% less diameter, p<0.05). Taken together, these data demonstrate that sEH‐inhibition attenuates vascular remodeling ‐ probably via alteration of SMC‐activity and augmentation of anti‐inflammatory, the vascular wall protecting EET‐effects.

Funding: German Research Foundation & J.W. Goethe‐University
Blockage of soluble epoxide hydrolase (sEH) enhances the anti‐inflammatory effects of aspirin and the 5‐lipoxygenase activation protein (FLAP) inhibitor MK886
Authors: Liu Junyan , Yang Jun , Inceoglu Bora , Ulu Arzu , Hwang Sung‐Hee , Hammock Bruce D
Published: June 2021

A previous study showed that co‐administration of a sEH inhibitor AUDA‐nBE enhances the antinociception of NSAIDs including indomethacin, celecoxib, or rofecoxib. We here demonstrate that co‐dosing of low dose of a novel potent sEH inhibitor t‐AUCB with aspirin or FLAP inhibitor MK886 produce significant beneficial effects on reversing hypotension in LPS‐challenged septic shock mouse model, and reducing the expression of inflammatory associated proteins (COX‐2 or 5‐LOX) and/or genes (TNF‐α and IL‐1β), as well as shifting the oxylipin metabolites. When administrated alone, aspirin decreased plasma TXB2 and PGE2 levels to 39 ± 14 %, and 47 ± 13 %, respectively, compared with the mice treated with LPS alone. When aspirin was used in a combination of low dose t‐AUCB, plasma TXB2 and PGE2 levels were dropped to 15 ± 10 % and 21 ± 5.0 %, respectively, versus LPS control. MK886 decreased plasma 5‐HETE levels to 41 ± 11 % versus LPS control. When MK886 was co‐administrated with low dose t‐AUCB, plasma 5‐HETE levels were dropped to 22 ± 2.7 % versus LPS control. Furthermore, targeted disruption of sEH gene produces the similar significant beneficial effects to pharmacological inhibition of sEH in LPS‐challenged septic shock mouse model. These observations show that blockage of sEH by both pharmacological intervention and gene knockout enhances the anti‐inflammatory effect of aspirin and FLAP inhibitors.
B‐type Natriuretic Peptide and EET Mediated Cardioprotection
Authors: Chaudhary Ketul , Batchu Sri Nagarjun , Das Dipankar , Suresh Mavanur , Graves Joan , Zeldin Darryl C , Seubert John M
Published: June 2021

B‐type natriuretic peptide (BNP) can attenuate ischemic‐reperfusion injury through natriuretic peptide receptor type‐A (NPR‐A). We investigated the role of BNP in epoxyeicosatrienoic acids(EET) mediated cardioprotection. Hearts from sEH null (KO), littermate controls(WT) and C57 mice were perfused in a Langendorff apparatus and subjected to 20/30min of ischemia followed by 40min of reperfusion. RT‐PCR analysis for preproBNP (Nppb) mRNA expression was performed in both naïve and isolated perfused hearts. Relative to naïve hearts, KO mice had a significant increase in Nppb mRNA expression following both preischemic perfusion (KO 5‐fold; WT 2‐fold) and postischemic reperfusion (KO 19‐fold; WT 7‐fold), correlating with improved postischemic left ventricular (LV) function (KO 52±3; WT 23.1±3%). KATP channel blocker, glibenclamide(1μM), and an EET antagonist, 14,15‐EEZE (1μM), significantly reduced the expression of mRNA in KO hearts compared to vehicle controls. Addition of exogenous BNP or EETs resulted in significant increases in LV functional recovery (BNP 59.2±13, EET 54.4±21, control 23.1±3%). BNP‐mediated protection was abolished with co‐administration of 14,15‐EEZE or A71915 (NPR‐A antagonist) (14,15‐EEZE 18.3±10, A71915 39.7±19%). However, EET‐mediated effects were not affected by A71915 (48.5±25%). Our results suggest a correlation between BNP and EET‐mediated cardioprotection.
A Water‐Soluble Formulation of HET0016 for the In‐Vivo Inhibition of 20‐HETE
Authors: Klamerus Megan Marie , Poloyac Samuel Mark , Rohan Lisa Cencia
Published: June 2021

Purpose: N‐Hydroxy‐N’‐(4‐butyl‐2‐methylphenyl)‐formamidine (HET0016) is a potent inhibitor of the cytochrome P450 enzymes responsible for the formation of the small vessel vasoconstrictor 20‐Hydroxyeicosatetraenoic acid (20‐HETE). However, HET0016 has a low aqueous solubility, limiting in vivo administration. The aim of this study was to use 2‐hydroxypropyl β‐cyclodextrin (2‐HPβCD) to create a water‐soluble formulation of HET0016.

Methods: The water solubility of HET0016 in both ddH2O and 15% 2‐HPβCD was determined. Rats then received 15% 2‐HPβCD or a formulation of HET0016 and 2‐HPβCD. Brain cortex, kidney and liver were subsequently analyzed for 20‐HETE content via HPLC/MS‐MS. Additional studies were also performed in order to maximize the amount of HET0016 and minimize the amount of 2‐HPβCD in the formulation.

Results: The solubility of HET0016 in ddH2O was found to be 34.2μg/mL compared to 452.7μg/mL in 15% 2‐HPβCD. 20‐HETE concentrations for the control group were 38.5, 189.2 and 835ng/g tissue in the brain cortex, kidney and liver, respectively. 20‐HETE concentrations in the same tissues for the experimental group were 9.8, 37.4 and 231.7ng/g tissue, respectively. Optimization of the HET0016:2‐HPβCD formulation showed that the greatest amount of HET0016 present in the smallest percentage of 2‐HPβCD was 1.3mg in 12.5%.

Conclusions: 2‐HPβCD increased the water solubility of HET0016 thirteen‐fold and also provided a water‐soluble formulation of this compound. When administered to rats, 20‐HETE formation was inhibited by 74.7% in the brain cortex, by 80.2% in the kidney and by 72.3% in the liver.

This work is supported by the NIH.
Topical Dual VEGF Receptor/Src Kinase Inhibitors as Potential Therapies for Ischemic Retinal Vascular Disorders
Authors: Mahesh Sankaranarayana Pillai , Virata Cyrus A , Chen Zoe , Cao Jon , Mak Chi Ching , McPherson Andrew , Noronha Glenn , Soll Richard
Published: June 2021

Purpose: Ischemic retinal vein occlusion (RVO) is a common retinal vascular disorder and carries a high risk of visual loss with few therapeutic options. We evaluated whether dual inhibition of VEGFR and down‐stream Src family tyrosine kinases using small molecules is a potential therapeutic intervention for ischemic retinal vascular disorders.

Methods: We examined two distinct chemical classes of dual VEGFR/Src kinase inhibitors (represented by compounds TG100801 and TG100948) in a rat model of RVO for their ability to impact retinal edema. Ischemic retinal vein occlusion was induced in rat retina by thrombosis of a single retinal vein using argon laser photocoagulation following injection of a photo‐sensitizer dye. Edema was quantified either using scanning optical fluorophotometry, measuring fluorescein leakage in to the retina, or by optical coherence tomography (OCT), measuring retinal thickness.

Results: Following topical administration, both the compounds achieved substantial levels in the retina and intraocular tissues with little to no systemic exposure. At 48 hrs post‐vein occlusion, both TG100801 and TG100948 successfully reduced edema compared to vehicle (by 47% with 0.3% TG100801, P= 0.002, and by 38% with 0.03% TG100948, P= 0.039). OCT scans also showed reduced retinal thickening as well as resolution of pockets of bullous edema.

Conclusions: As VEGF plays a key role in the edema and neovascularization which develop in the ischemic retina, we propose that multi‐targeted inhibitors of the VEGF signaling cascade represent promising therapies for these vascular disorders. Moreover, as current forms of therapy require invasive delivery techniques (intravitreal injections), compounds such as TG100801 and TG100948, which can be topically delivered, offer additional therapeutic advantages.
Scavenger receptor BI prevents septic death
Authors: Li Xiang‐An , Guo Ling , Song Zhiqing , Smart Eric J
Published: June 2021

Scavenger receptor BI (SR‐BI) is a HDL receptor which regulates plasma HDL cholesterol levels by selectively uptake cholesterol ester from HDL. We recently reported a novel function of SR‐BI namely protection against endotoxin induced animal death. Using a lipopolysaccharides (LPS) induced endotoxic animal model, we found that LPS challenge induced 90% fatality of SR‐BI null mice while all the wild type littermates survived, indicating that SR‐BI plays a pivotal role in protection against LPS toxicity in vivo. In the current studies, we determined whether SR‐BI plays a protective role in sepsis using an established septic animal model (cecum ligation and puncture, CLP). We demonstrate that CLP treatment induced 100% fatality of SR‐BI null mice within three days (n=20) whereas only 21% fatality of wild type littermates (n=27) and 36% fatality of heterozygous littermates (n=30). Our findings clearly demonstrate that SR‐BI is a critical protective modulator in sepsis in mice, which may reveal a novel target for the intervention of sepsis.

(Supported by grants from American Heart Association 0530241N and from the NIH 2P20RR015592)