Perivascular adipose tissue remodeling impairs vasoreactivity in thermoneutral-housed rats.

Objective: Vascular pathology, characterized by impaired vasoreactivity and mitochondrial respiration, differs between the sexes. Housing rats under thermoneutral (TN) conditions causes vascular dysfunction and perturbed metabolism. We hypothesized that perivascular adipose tissue (PVAT), a vasoregulatory adipose depot with brown adipose tissue (BAT) phenotype, remodels to a white adipose (WAT) phenotype in rats housed at TN, driving diminished vasoreactivity in a sex-dependent manner. Methods: Male and female Wistar rats were housed at either room temperature (RT) or TN. Endpoints included changes in PVAT morphology, vasoreactivity in vessels with intact PVAT or transferred to PVAT of the oppositely-housed animal, vessel stiffness, vessel mitochondrial respiration and cellular signaling. Results: Remodeling of PVAT was observed in rats housed at TN; animals in this environment showed PVAT whitening and displayed diminished aortae vasodilation (p<0.05), different between the sexes. Juxtaposing PVAT from RT rats onto aortae from TN rats in females corrected vasodilation (p<0.05); this did not occur in males. In aortae of all animals housed at TN, mitochondrial respiration was significantly diminished in lipid substrate experiments (p<0.05), and there was significantly less expression of peNOS (p<0.001). Conclusions: These data are consistent with TN-induced remodeling of PVAT, notably associated with sex-specific blunting of vasoreactivity, diminished mitochondrial respiration, and altered cellular signaling.

Improving Employee Safety Through a Comprehensive Patient Behavioral Program.

BACKGROUND: Health care workers in the United States are facing increasing rates of exposure to aggressive behavior, resulting in an increase in employee injuries related specifically to patient behavioral events. By leveraging interprofessional collaboration and system-level innovation, we aimed to reduce the rate of employee injuries related to patient behavioral events at a children's hospital by 50% over a 3-year period. METHODS: An interdisciplinary quality improvement team comprising physicians, behavior analysts, nursing, and other key stakeholders developed a comprehensive behavior program in our children's hospital. The team developed 5 key pillars: aggression mitigation tools, clinical resources, advanced training, screening and management, and behavior emergency response. The outcome measure was rate of reported employee safety events related to patient behavioral events. This was tracked via prospective time series analysis statistical process control chart using established rules to detect special cause variation. RESULTS: The average rate of employee injuries resulting from patient behavioral events decreased from 0.96 to 0.39 per 1000 adjusted patient-days, with special cause variation observed on a statistical process control U-chart. This improvement has been sustained for 16 months. Staff members who experienced injuries included nurses and patient technicians, with common antecedents to injuries including medical interventions or patient requests that could not be safely met. CONCLUSIONS: A unified and multimodal system aimed to address pediatric patient behavioral events can reduce employee injuries and foster a culture of employee safety in the pediatric inpatient setting.

Gluten cross contact in oats: retrospective database analysis 2011 to 2023.

It is long-established that oats are at substantial risk for cross contact with gluten-containing grain. Specially processed gluten-free oats, whether purity protocol or mechanically/optically sorted, made it possible for this grain to be included in a gluten-free diet in the U.S. Gluten Free Watchdog (GFWD) (Manchester, Massachusetts, United States) has been assessing the gluten content of labeled gluten-free foods since 2011. In 2022, there was an apparent increase in the number of oat products testing with quantifiable gluten at or above 5 mg/kg or parts per million (ppm). The purpose of the present study was to assess the levels of gluten in foods containing oats to determine if there were any trends. In this retrospective database analysis, GFWD product test reports from April 2011 to May 1, 2023 were searched using the term "oat." The search identified 213 individual packages of food that contained the word "oat" in the ingredients list. The test results for these packages of food were reviewed. Of these, 24 (11%) tested with quantifiable gluten greater than or equal to 5 mg/kg (ppm). The percentage of oats testing with quantifiable gluten varied per year but spiked in 2022 at 35%. It is not possible to know for certain what caused this increase. The drought during the oat growing season of 2021 could be a major factor. This drought impacted oat crops in both the US and Canada and led to one of the worst oat crops going back over 150 years. One limitation of this study is that it was a retrospective analysis. Different numbers of oat products were tested each year and these were often different brands of oats and different oat formulations. To assess the level of gluten cross contact in oats going forward a much larger prospective study should be conducted.

(-)-Epicatechin Reverses Glucose Intolerance in Rats Housed at Thermoneutrality.

Diabetes is a life-threatening and debilitating disease with pathological hallmarks, including glucose intolerance and insulin resistance. Plant compounds are a source of novel and effective therapeutics, and the flavonoid (-)-epicatechin, common to popular foods worldwide, has been shown to improve carbohydrate metabolism in both clinical studies and preclinical models. We hypothesized that (-)-epicatechin would alleviate thermoneutral housing-induced glucose intolerance. Male rats were housed at either thermoneutral (30 °C) or room temperature (24 °C) for 16 weeks and gavaged with either 1 mg/kg body weight or vehicle for the last 15 days before sacrifice. Rats housed at thermoneutrality had a significantly elevated serum glucose area under the curve (p < 0.05) and reduced glucose-mediated insulin secretion. In contrast, rats at thermoneutrality treated with (-)-epicatechin had improved glucose tolerance and increased insulin secretion (p < 0.05). Insulin tolerance tests revealed no differences in insulin sensitivity in any of the four groups. Pancreatic immunohistochemistry staining showed significantly greater islet insulin positive cells in animals housed at thermoneutrality. In conclusion, (-)-epicatechin improved carbohydrate tolerance via increased insulin secretion in response to glucose challenge without a change in insulin sensitivity.

Thermoneutrality induces vascular dysfunction and impaired metabolic function in male Wistar rats: a new model of vascular disease.

OBJECTIVE: Cardiovascular disease is of paramount importance, yet there are few relevant rat models to investigate its pathology and explore potential therapeutics. Housing at thermoneutral temperature (30 °C) is being employed to humanize metabolic derangements in rodents. We hypothesized that housing rats in thermoneutral conditions would potentiate a high-fat diet, resulting in diabetes and dysmetabolism, and deleteriously impact vascular function, in comparison to traditional room temperature housing (22 °C). METHODS: Male Wistar rats were housed at either room temperature or thermoneutral temperatures for 16 weeks on either a low or high-fat diet. Glucose and insulin tolerance tests were conducted at the beginning and end of the study. At the study's conclusion, vasoreactivity and mitochondrial respiration of aorta and carotid were conducted. RESULTS: We observed diminished vasodilation in vessels from thermoneutral rats ( P  < 0.05), whereas high-fat diet had no effect. This effect was also observed in endothelium-denuded aorta in thermoneutral rats ( P  < 0.05). Vasoconstriction was significantly elevated in aorta of thermoneutral rats ( P  < 0.05). Diminished nitric oxide synthase activity and nitrotyrosine, and elevated glutathione activity were observed in aorta from rats housed under thermoneutral conditions, indicating a climate of lower nitric oxide and excess reactive oxygen species in aorta. Thermoneutral rat aorta also demonstrated less mitochondrial respiration with lipid substrates compared with the controls ( P  < 0.05). CONCLUSION: Our data support that thermoneutrality causes dysfunctional vasoreactivity, decreased lipid mitochondrial metabolism, and modified cellular signaling. These are critical observations as thermoneutrality is becoming prevalent for translational research models. This new model of vascular dysfunction may be useful for dissection of targetable aspects of cardiovascular disease and is a novel and necessary model of disease.

Lentils and Gluten Cross Contact.

Lentils are naturally gluten-free and are recommended for people with celiac disease and other gluten-related disorders. However, like oats, they appear to be at a heightened risk of cross contact with gluten-containing grains. The purpose of this study was to spot check for the presence of errant gluten-containing grains in a variety of brands of lentils purchased in 2021. Twenty-five bags of different dry lentil products representing 24 brands were purchased online and at various grocery stores. Each bag of lentils was individually hand sorted. Two of the 25 packages of lentils contained errant gluten-containing grains. One 16-ounce (454 g) bag contained a grain of wheat. Another 16-ounce (454 g) bag contained a grain of wheat and a grain of barley. For a product to be considered gluten-free in the United States, it must contain <20 mg of gluten per kilogram (or 20 parts per million of gluten). A product at the 20-ppm level of gluten should contain no more than 2 intact gluten-containing grains per kilogram or 35.27 ounces (1,000 g) of food (or 1 intact gluten-containing grain in 17.64 ounces [500 g] of food). Based on these calculations, a 16-ounce (454 g) bag of lentils containing 1 intact gluten-containing grain would not be considered gluten-free. Lentils are at risk of cross contact with gluten-containing grain. Consumers should continue to sort through lentils removing foreign grain, and rinse sorted lentils under running water to remove grain dust before cooking.

Maternal obesity causes fetal cardiac hypertrophy and alters adult offspring myocardial metabolism in mice.

Obesity in pregnant women causes fetal cardiac dysfunction and increases offspring cardiovascular disease risk, but its effect on myocardial metabolism is unknown. We hypothesized that maternal obesity alters fetal cardiac expression of metabolism-related genes and shifts offspring myocardial substrate preference from glucose towards lipids. Female mice were fed control or obesogenic diets before and during pregnancy. Fetal hearts were studied in late gestation (embryonic day (E) 18.5; term ≈ E21), and offspring were studied at 3, 6, 9 or 24 months postnatally. Maternal obesity increased heart weight and peroxisome proliferator activated receptor gamma (Pparg) expression in female and male fetuses and caused left ventricular diastolic dysfunction in the adult offspring. Cardiac dysfunction worsened progressively with age in female, but not male, offspring of obese dams, in comparison to age-matched control animals. In 6-month-old offspring, exposure to maternal obesity increased cardiac palmitoyl carnitine-supported mitochondrial respiration in males and reduced myocardial 18 F-fluorodeoxyglucose uptake in females. Cardiac Pparg expression remained higher in adult offspring of obese dams than control dams and was correlated with contractile and metabolic function. Maternal obesity did not affect cardiac palmitoyl carnitine respiration in females or 18 F-fluorodeoxyglucose uptake in males and did not alter cardiac 3 H-oleic acid uptake, pyruvate respiration, lipid content or fatty acid/glucose transporter abundance in offspring of either sex. The results support our hypothesis and show that maternal obesity affects offspring cardiac metabolism in a sex-dependent manner. Persistent upregulation of Pparg expression in response to overnutrition in utero might underpin programmed cardiac impairments mechanistically and contribute to cardiovascular disease risk in children of women with obesity. KEY POINTS: Obesity in pregnant women causes cardiac dysfunction in the fetus and increases lifelong cardiovascular disease risk in the offspring. In this study, we showed that maternal obesity in mice induces hypertrophy of the fetal heart in association with altered expression of genes related to nutrient metabolism. Maternal obesity also alters cardiac metabolism of carbohydrates and lipids in the adult offspring. The results suggest that overnutrition in utero might contribute to increased cardiovascular disease risk in children of women with obesity.

Mitigating Pediatric Inpatient Aggression: A Quality Improvement Initiative.

BACKGROUND: Patient aggression in the health care workplace has increased significantly, and the impact of workplace violence can be profound, including psychological trauma and lost productivity. We suspect these safety events are often unreported, leading to missed opportunities to design interventions to reduce harm. The primary aim of the interdisciplinary quality improvement team was to increase staff reporting of safety events utilizing our event reporting system related to the care of verbally and/or physically aggressive pediatric patients by 10% over a 12-month period. METHODS: An interdisciplinary quality improvement team addressed existing gaps in the care of pediatric inpatients with escalating behavior. Interventions included a survey of staff knowledge, use of the care guideline for management, updates to the electronic medical record, patient aggression screening tool, an electronic order set, and an online education module. The primary outcome measure was the number of reported staff safety events related to the care of aggressive patients. Compliance with the use of the pediatric aggression risk screening tool was tracked as a process measure. RESULTS: The reporting of safety events related to the care of aggressive patients increased from just <1.0 events per 1000 patient days to 3.0 with special cause variation observed on a statistical process control chart. The compliance with the use of the pediatric aggression risk screening tool improved during the time of the project, nearing 90%. CONCLUSIONS: A variety of interventions aimed to address pediatric inpatient aggression can improve the reporting of events related to workplace violence and foster a culture of employee safety.

(-)-Epicatechin Improves Vasoreactivity and Mitochondrial Respiration in Thermoneutral-Housed Wistar Rat Vasculature.

Cardiovascular disease (CVD) is a global health concern. Vascular dysfunction is an aspect of CVD, and novel treatments targeting vascular physiology are necessary. In the endothelium, eNOS regulates vasodilation and mitochondrial function; both are disrupted in CVD. (−)-Epicatechin, a botanical compound known for its vasodilatory, eNOS, and mitochondrial-stimulating properties, is a potential therapy in those with CVD. We hypothesized that (−)-epicatechin would support eNOS activity and mitochondrial respiration, leading to improved vasoreactivity in a thermoneutral-derived rat model of vascular dysfunction. We housed Wistar rats at room temperature or in thermoneutral conditions for a total of 16 week and treated them with 1mg/kg body weight (−)-epicatechin for 15 day. Vasoreactivity, eNOS activity, and mitochondrial respiration were measured, in addition to the protein expression of upstream cellular signaling molecules including AMPK and CaMKII. We observed a significant improvement of vasodilation in those housed in thermoneutrality and treated with (−)-epicatechin (p < 0.05), as well as dampened mitochondrial respiration (p < 0.05). AMPK and CaMKIIα and ß expression were lessened with (−)-epicatechin treatment in those housed at thermoneutrality (p < 0.05). The opposite was observed with animals housed at room temperature supplemented with (−)-epicatechin. These data illustrate a context-dependent vascular response to (−)-epicatechin, a candidate for CVD therapeutic development.

Identification of kidney injury released circulating osteopontin as causal agent of respiratory failure.

Tissue injury can drive secondary organ injury; however, mechanisms and mediators are not well understood. To identify interorgan cross-talk mediators, we used acute kidney injury (AKI)-induced acute lung injury (ALI) as a clinically important example. Using kidney and lung single-cell RNA sequencing after AKI in mice followed by ligand-receptor pairing analysis across organs, kidney ligands to lung receptors, we identify kidney-released circulating osteopontin (OPN) as a novel AKI-ALI mediator. OPN release from kidney tubule cells triggered lung endothelial leakage, inflammation, and respiratory failure. Pharmacological or genetic OPN inhibition prevented AKI-ALI. Transplantation of ischemic wt kidneys caused AKI-ALI, but not of ischemic OPN-global knockout kidneys, identifying kidney-released OPN as necessary interorgan signal to cause AKI-ALI. We show that OPN serum levels are elevated in patients with AKI and correlate with kidney injury. Our results demonstrate feasibility of using ligand-receptor analysis across organs to identify interorgan cross-talk mediators and may have important therapeutic implications in human AKI-ALI and multiorgan failure.

Gluten-Free Foods Cooked in Shared Fryers With Wheat: A Pilot Study Assessing Gluten Cross Contact.

Introduction: Consumers with celiac disease are discouraged from eating fried foods cooked in shared fryers with wheat-containing foods at restaurants based on presumed gluten exposure. The purpose of the present study is to assess gluten levels of fries free of gluten-containing ingredients cooked in shared fryers with wheat. Methods: 20 orders of fries were purchased from 10 different restaurants. Restaurants confirmed that fries and oil were free of gluten-containing ingredients. All restaurants confirmed that their fryers were used to cook wheat-containing foods. Fries were sent to Bia Diagnostics and tested in 1-gram duplicates using the R7001 sandwich R5 ELISA and the R7021 competitive R5 ELISA. A microwave control also was run. Results: The sandwich ELISA found gluten in 9/20 fry orders (7 to > 80 ppm). The competitive ELISA found gluten in 3/20 fry orders (14 to > 270 ppm). In the microwave control (60-ppm gluten mixture of wheat flour and canola oil), the unheated mixture tested at a mean level of 64 ppm gluten using the sandwich ELISA and 137 ppm gluten using the competitive ELISA. The mixture heated to 190°C tested at a mean level of 55 ppm gluten using the sandwich ELISA and < 10 ppm and 16 ppm gluten using the competitive ELISA. Discussion: Based on test results, 25% of fry orders would not be considered gluten-free. Summary: Gluten cross contact may occur when gluten-free foods are cooked in shared fryers with wheat. ELISAs may underperform when analyzing for gluten that has been heated.

(-)-Epicatechin Modulates Mitochondrial Redox in Vascular Cell Models of Oxidative Stress.

Diabetes mellitus affects 451 million people worldwide, and people with diabetes are 3-5 times more likely to develop cardiovascular disease. In vascular tissue, mitochondrial function is important for vasoreactivity. Diabetes-mediated generation of excess reactive oxygen species (ROS) may contribute to vascular dysfunction via damage to mitochondria and regulation of endothelial nitric oxide synthase (eNOS). We have identified (-)-epicatechin (EPICAT), a plant compound and known vasodilator, as a potential therapy. We hypothesized that mitochondrial ROS in cells treated with antimycin A (AA, a compound targeting mitochondrial complex III) or high glucose (HG, global perturbation) could be normalized by EPICAT, and correlate with improved mitochondrial dynamics and cellular signaling. Human umbilical vein endothelial cells (HUVEC) were treated with HG, AA, and/or 0.1 or 1.0 µM of EPICAT. Mitochondrial and cellular superoxide, mitochondrial respiration, and cellular signaling upstream of mitochondrial function were assessed. EPICAT at 1.0 µM significantly attenuated mitochondrial superoxide in HG-treated cells. At 0.1 µM, EPICAT nonsignificantly increased mitochondrial respiration, agreeing with previous reports. EPICAT significantly increased complex I expression in AA-treated cells, and 1.0 µM EPICAT significantly decreased mitochondrial complex V expression in HG-treated cells. No significant effects were seen on either AMPK or eNOS expression. Our study suggests that EPICAT is useful in mitigating moderate ROS concentrations from a global perturbation and may modulate mitochondrial complex activity. Our data illustrate that EPICAT acts in the cell in a dose-dependent manner, demonstrating hormesis.

Acute estradiol treatment reduces skeletal muscle protein breakdown markers in early- but not late-postmenopausal women.

OBJECTIVES: Menopause and decline in estradiol (E2) may contribute to sarcopenia (i.e., age-related decline in muscle mass and strength) in women. E2 may directly impact skeletal muscle protein breakdown via estrogen receptor (ER) signaling, primarily ERα. It is not yet known whether: 1) E2 regulates pathways of skeletal muscle protein breakdown; 2) E2-mediated changes in protein breakdown markers are associated with ERα activation and insulin sensitivity; and 3) the effects of E2 on protein breakdown markers differ by increasing time since menopause. STUDY DESIGN: We studied 27 women who were ≤6 years past menopause (early postmenopausal, EPM; n = 13) or ≥10 years past menopause (late postmenopausal, LPM; n = 14). Fasted skeletal muscle samples were collected following 1 week of transdermal E2 or placebo treatment in a randomized cross-over design. MAIN OUTCOME MEASURES: We analyzed for cytosolic protein content of the: 1) structural proteins myosin heavy chain (MHC) and tropomyosin; and 2) protein regulatory markers: protein kinase B (Akt), muscle-specific ring finger protein1 (MuRF1), atrogin1, and forkhead box O3 (FOXO3) using Western blot. RESULTS: In response to acute E2, FOXO3 activation (dephosphorylation) and MuRF1 protein expression decreased in EPM but increased in LPM women (p < 0.05). ERα activation was not associated with these protein breakdown markers, but FOXO3 activation tended to be inversely correlated (r = -0.318, p = 0.065) to insulin sensitivity. CONCLUSIONS: These preliminary studies suggest the effects of E2 on skeletal muscle protein breakdown markers were dependent on time since menopause, which is consistent with our previous study on insulin sensitivity.

Elevated plasma homocysteine and cysteine are associated with endothelial dysfunction across menopausal stages in healthy women.

Hyperhomocysteinemia is associated with endothelial dysfunction and increased cardiovascular disease (CVD). We determined whether elevated homocysteine (Hcy) and markers of Hcy metabolism were associated with the previously reported endothelial dysfunction across stages of the menopause transition. Brachial artery flow-mediated dilation (FMD) and plasma concentrations of Hcy, cysteine, and methionine were measured in healthy women (n = 128) 22-70 yr of age categorized as premenopausal (n = 35), perimenopausal (early: n = 16; late: n = 21), and postmenopausal (early: n = 21; late: n = 35). Dietary intake of micronutrients involved in Hcy metabolism (e.g., vitamins B6, B12, folate) was assessed in a subpopulation of women. Hcy and cysteine concentrations were progressively higher, and methionine was progressively lower across menopausal stages (all P < 0.005). The higher Hcy and cysteine concentrations correlated with lower circulating estradiol levels (r = -0.49 and -0.50, respectively, both P < 0.001). FMD was inversely correlated with Hcy (r = -0.25, P = 0.004) and cysteine (r = -0.39, P < 0.001) and positively correlated with methionine concentrations (r = 0.25, P = 0.005). Dietary intake of vitamins B6 and B12 (both P < 0.05) were lower in postmenopausal women. Vitamin B12 intake correlated with FMD (r = 0.22, P = 0.006). These data suggest that declines in estradiol across stages of the menopause transition may lead to elevations in Hcy and cysteine that may contribute to endothelial dysfunction in postmenopausal women. Future studies should examine whether targeting Hcy metabolism during the perimenopausal to early postmenopausal period with interventions, including diet, attenuates or reverses the decline in endothelial function in women. NEW & NOTEWORTHY Declines in circulating estradiol across the stages of the menopausal transition may lead to elevations in Hcy and cysteine concentrations that may contribute to endothelial dysfunction. Abnormalities in the Hcy metabolic pathways, possibly related to dietary deficiencies of vitamins B12 and B6 and folate, may contribute to elevations in Hcy and cysteine concentrations. Findings also suggest that higher cysteine levels may be more damaging to the vascular endothelium than Hcy.

Glucagon-like peptide-1 receptor antagonism impairs basal exercise capacity and vascular adaptation to aerobic exercise training in rats.

Cardiorespiratory fitness (CRF) inversely predicts cardiovascular (CV) mortality and CRF is impaired in people with type 2 diabetes (T2D). Aerobic exercise training (ET) improves CRF and is associated with decreased risk of premature death in healthy and diseased populations. Understanding the mechanisms contributing to ET adaptation may identify targets for reducing CV mortality of relevance to people with T2D. The antihyperglycemic hormone glucagon-like peptide-1 (GLP-1) influences many of the same pathways as exercise and may contribute to CV adaptation to ET. We hypothesized that GLP-1 is necessary for adaptation to ET. Twelve-week-old male Wistar rats were randomized (n = 8-12/group) to receive PBS or GLP-1 receptor antagonist (exendin 9-39 (Ex(9-39)) via osmotic pump for 4 weeks ± ET. CRF was greater with ET (P < 0.01). Ex(9-39) treatment blunted CRF in both sedentary and ET rats (P < 0.001). Ex(9-39) attenuated acetylcholine-mediated vasodilation, while this response was maintained with Ex(9-39)+ET (P = 0.04). Aortic stiffness was greater with Ex(9-39) (P = 0.057) and was made worse when Ex(9-39) was combined with ET (P = 0.004). Ex vivo aortic vasoconstriction with potassium and phenylephrine was lower with Ex(9-39) (P < 0.0001). Carotid strain improved with PBS + ET but did not change in the Ex(9-39) rats with ET (P < 0.0001). Left ventricular mitochondrial respiration was elevated with Ex(9-39) (P < 0.02). GLP-1 receptor antagonism impairs CRF with and without ET, attenuates the vascular adaptation to ET, and elevates cardiac mitochondrial respiration. These data suggest that GLP-1 is integral to the adaptive vascular response to ET.

Varicella zoster virus infection of human fetal lung cells alters mitochondrial morphology.

Varicella zoster virus (VZV) is a ubiquitous alphaherpesvirus that establishes latency in ganglionic neurons throughout the neuraxis after primary infection. Here, we show that VZV infection induces a time-dependent significant change in mitochondrial morphology, an important indicator of cellular health, since mitochondria are involved in essential cellular functions. VZV immediate-early protein 63 (IE63) was detected in mitochondria-rich cellular fractions extracted from infected human fetal lung fibroblasts (HFL) by Western blotting. IE63 interacted with cytochrome c oxidase in bacterial 2-hybrid analyses. Confocal microscopy of VZV-infected HFL cells at multiple times after infection revealed the presence of IE63 in the nucleus, mitochondria, and cytoplasm. Our data provide the first evidence that VZV infection induces alterations in mitochondrial morphology, including fragmentation, which may be involved in cellular damage and/or death during virus infection.

Differential Mitochondrial Adaptation in Primary Vascular Smooth Muscle Cells from a Diabetic Rat Model.

Diabetes affects more than 330 million people worldwide and causes elevated cardiovascular disease risk. Mitochondria are critical for vascular function, generate cellular reactive oxygen species (ROS), and are perturbed by diabetes, representing a novel target for therapeutics. We hypothesized that adaptive mitochondrial plasticity in response to nutrient stress would be impaired in diabetes cellular physiology via a nitric oxide synthase- (NOS-) mediated decrease in mitochondrial function. Primary smooth muscle cells (SMCs) from aorta of the nonobese, insulin resistant rat diabetes model Goto-Kakizaki (GK) and the Wistar control rat were exposed to high glucose (25 mM). At baseline, significantly greater nitric oxide evolution, ROS production, and respiratory control ratio (RCR) were observed in GK SMCs. Upon exposure to high glucose, expression of phosphorylated eNOS, uncoupled respiration, and expression of mitochondrial complexes I, II, III, and V were significantly decreased in GK SMCs (p < 0.05). Mitochondrial superoxide increased with high glucose in Wistar SMCs (p < 0.05) with no change in the GK beyond elevated baseline concentrations. Baseline comparisons show persistent metabolic perturbations in a diabetes phenotype. Overall, nutrient stress in GK SMCs caused a persistent decline in eNOS and mitochondrial function and disrupted mitochondrial plasticity, illustrating eNOS and mitochondria as potential therapeutic targets.

Raw and processed microscope images of fixed cells at baseline and following various experimental perturbations.

The data included in this article comprise raw and processed images of fixed cells at baseline and subjected to various experimental perturbations. This dataset includes images of HUVEC cells fixed and subsequently incubated at either 37 °C or room temperature, primary rat vascular smooth muscle cells exposed to 25 mM glucose, and SH-SY5Y neurons exposed to hydrogen peroxide. Raw images appear exactly as they were captured on the microscope, while processed images show the binarization provided by software used for measurements of mitochondrial morphology. For in-depth discussion of the experiments and computational methods pertaining to this data, please refer to the corresponding research article titled "Fully automated software for quantitative measurements of mitochondrial morphology" (McClatchey et al., in press) [1].

Fully automated software for quantitative measurements of mitochondrial morphology.

Mitochondria undergo dynamic changes in morphology in order to adapt to changes in nutrient and oxygen availability, communicate with the nucleus, and modulate intracellular calcium dynamics. Many recent papers have been published assessing mitochondrial morphology endpoints. Although these studies have yielded valuable insights, contemporary assessment of mitochondrial morphology is typically subjective and qualitative, precluding direct comparison of outcomes between different studies and likely missing many subtle effects. In this paper, we describe a novel software technique for measuring the average length, average width, spatial density, and intracellular localization of mitochondria from a fluorescent microscope image. This method was applied to distinguish baseline characteristics of Human Umbilical Vein Endothelial Cells (HUVECs), primary Goto-Kakizaki rat aortic smooth muscle cells (GK SMCs), primary Wistar rat aortic smooth muscle cells (Wistar SMCs), and SH-SY5Ys (human neuroblastoma cell line). Consistent with direct observation, our algorithms found SH-SY5Ys to have the greatest mitochondrial density, while HUVECs were found to have the longest mitochondria. Mitochondrial morphology responses to temperature, nutrient, and oxidative stressors were characterized to test algorithm performance. Large morphology changes recorded by the software agreed with direct observation, and subtle but consistent morphology changes were found that would not otherwise have been detected. Endpoints were consistent between experimental repetitions (R=0.93 for length, R=0.93 for width, R=0.89 for spatial density, and R=0.74 for localization), and maintained reasonable agreement even when compared to images taken with compromised microscope resolution or in an alternate imaging plane. These results indicate that the automated software described herein allows quantitative and objective characterization of mitochondrial morphology from fluorescent microscope images.