Differences in vasomotor function of mesenteric arteries between Ossabaw minipigs with predisposition to metabolic syndrome and Göttingen minipigs.

Metabolic syndrome predisposes and contributes to the development and progression of atherosclerosis. The minipig strain "Ossabaw" is characterized by a predisposition to develop metabolic syndrome. We compared vasomotor function in Ossabaw minipigs before they developed their diseased phenotype to that of Göttingen minipigs without such genetic predisposition. Mesenteric arteries of adult Ossabaw and Göttingen minipigs were dissected postmortem and mounted on a myograph for isometric force measurements. Maximal vasoconstriction to potassium chloride (KClmax) was induced. Cumulative concentration-response curves were determined in response to norepinephrine. Endothelium-dependent (with carbachol) and endothelium-independent (with nitroprusside) vasodilation were analyzed after preconstriction by norepinephrine. In a bioinformatic analysis, variants/altered base pairs within genes associated with cardiovascular disease were analyzed. KClmax was similar between the minipig strains (15.6 ± 6.7 vs. 14.1 ± 3.4 ΔmN). Vasoconstriction in response to norepinephrine was more pronounced in Ossabaw than in Göttingen minipigs (increase of force to 143 ± 48 vs. 108 ± 38% of KClmax). Endothelium-dependent and endothelium-independent vasodilation were less pronounced in Ossabaw than in Göttingen minipigs (decrease of force to 46.4 ± 29.6 vs. 16.0 ± 18.4% and to 36.7 ± 25.2 vs. 2.3 ± 3.7% of norepinephrine-induced preconstriction). Vasomotor function was not different between the sexes. More altered base pairs/variants were identified in Ossabaw than in Göttingen minipigs for the exon encoding adrenoceptor-α1A. Vasomotor function in lean Ossabaw minipigs is shifted toward vasoconstriction and away from vasodilation in comparison with Göttingen minipigs, suggesting a genetic predisposition for vascular dysfunction and atherosclerosis in Ossabaw minipigs. Thus, Ossabaw minipigs may be a better model for human cardiovascular disease than Göttingen minipigs.NEW & NOTEWORTHY Animal models with a predisposition to metabolic syndrome and atherosclerosis are attracting growing interest for translational research, as they may better mimic the variability of patients with cardiovascular disease. In Ossabaw minipigs, with a polygenic predisposition to metabolic syndrome, but without the diseased phenotype, vasoconstriction is more and vasodilation is less pronounced in mesenteric arteries than in Göttingen minipigs. Ossabaw minipigs may be a more suitable model of human cardiovascular disease.

Remote ischemic conditioning in Ossabaw minipigs induces the release of humoral cardioprotective triggers, but the myocardium does not respond with reduced infarct size.

Ischemic preconditioning (IPC; brief cycles of coronary occlusion/reperfusion) is operative in all species tested so far and reduces infarct size through the release of trigger molecules and activation of signal transducer and activator of transcription (STAT)3 in pigs. We have recently demonstrated that IPC failed to protect Ossabaw minipigs, which had a genetic predisposition to, but not yet established a metabolic syndrome, from infarction and did not activate STAT3. We now subjected Ossabaw minipigs to remote ischemic conditioning (RIC; 4 × 5 min/5 min bilateral hindlimb ischemia-reperfusion) and analyzed the release of cardioprotective triggers into the circulation with the aim to distinguish whether IPC failed to stimulate trigger release or to activate intracellular signaling cascades upstream of STAT3. RIC or a placebo protocol, respectively, was induced in anesthetized pigs before 60 min/180 min coronary occlusion/reperfusion. Plasma, prepared from Ossabaw minipigs after RIC or placebo, was infused into isolated rat hearts subjected to 30 min/120 min global ischemia-reperfusion. In the Ossabaw minipigs, RIC did not reduce infarct size (49.5 ± 12.1 vs. 56.0 ± 11.8% of area at risk with placebo), and STAT3 was not activated. In isolated rat hearts, infusion of RIC plasma reduced infarct size (19.7 ± 6.7 vs. 33.2 ± 5.5% of ventricular mass with placebo) and activated STAT3. Pretreatment of rat hearts with the STAT3 inhibitor stattic abrogated such infarct size reduction and STAT3 activation. In conclusion, Ossabaw minipigs release cardioprotective triggers in response to RIC into the circulation, and lack of cardioprotection is attributed to myocardial nonresponsiveness.NEW & NOTEWORTHY Ischemic conditioning reduces myocardial infarct size in all species tested so far. In the present study, we used Ossabaw minipigs that had a genetic predisposition to, but not yet established a metabolic syndrome. In these pigs, remote ischemic conditioning (RIC) induced the release of cardioprotective triggers but did not reduce infarct size. Transfer of their plasma, however, reduced infarct size in isolated recipient rat hearts, along with signal transducer and activator of transcription (STAT)3 activation.

Non-responsiveness to cardioprotection by ischaemic preconditioning in Ossabaw minipigs with genetic predisposition to, but without the phenotype of the metabolic syndrome.

The translation of successful preclinical and clinical proof-of-concept studies on cardioprotection to the benefit of patients with reperfused acute myocardial infarction has been difficult so far. This difficulty has been attributed to confounders which patients with myocardial infarction typically have but experimental animals usually not have. The metabolic syndrome is a typical confounder. We hypothesised that there may also be a genuine non-responsiveness to cardioprotection and used Ossabaw minipigs which have the genetic predisposition to develop a diet-induced metabolic syndrome, but before they had developed the diseased phenotype. Using a prospective study design, a reperfused acute myocardial infarction was induced in 62 lean Ossabaw minipigs by 60 min coronary occlusion and 180 min reperfusion. Ischaemic preconditioning by 3 cycles of 5 min coronary occlusion and 10 min reperfusion was used as cardioprotective intervention. Ossabaw minipigs were stratified for their single nucleotide polymorphism as homozygous for valine (V/V) or isoleucine (I/I)) in the γ-subunit of adenosine monophosphate-activated protein kinase. Endpoints were infarct size and area of no-reflow. Infarct size (V/V: 54 ± 8, I/I: 54 ± 13% of area at risk, respectively) was not reduced by ischaemic preconditioning (V/V: 55 ± 11, I/I: 46 ± 11%) nor was the area of no-reflow (V/V: 57 ± 18, I/I: 49 ± 21 vs. V/V: 57 ± 21, I/I: 47 ± 21% of infarct size). Bioinformatic comparison of the Ossabaw genome to that of Sus scrofa and Göttingen minipigs identified differences in clusters of genes encoding mitochondrial and inflammatory proteins, including the janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. The phosphorylation of STAT3 at early reperfusion was not increased by ischaemic preconditioning, different from the established STAT3 activation by cardioprotective interventions in other pig strains. Ossabaw pigs have not only the genetic predisposition to develop a metabolic syndrome but also are not amenable to cardioprotection by ischaemic preconditioning.

Introduction to ion transport and membrane interactions in vascular health and disease.

Cardiovascular disease is on the rise, partially due to the continued increase in metabolic syndrome. Advances in basic research on vascular ion transport have the potential to provide targets for therapeutic interventions. Vascular specificity, which includes different vascular beds having different characteristics and the macro- vs. microvasculature, is a vitally important variable in characterization of ion transport. At the cellular level, targeted fluorescent biosensors for Ca2+, super-resolution microscopy, and organelle patch clamp electrophysiology enable more detailed studies. The "MetS/diabetes milieu" includes increased and decreased insulin, and increased glucose, increased LDL/HDL cholesterol and triglycerides, and increased blood pressure. The duration and severity of MetS/diabetes components certainly affect the vascular phenotype and ion transport and membrane interactions. A combination of in vivo animal models and in vitro cell models to study ion transport in MetS/diabetes conditions is optimal. Gene editing and selective pharmacological tools should be used after or in conjunction with characterization of ion transport in vascular health and disease phenotypes. This is critical to determining the causal role of Ca2+ signaling in modulation of vascular phenotype. The ion transport and membrane interactions that are measured are typically only a snapshot in time in these dynamic processes occurring over the progression of health and disease. It is imperative that this concept be considered in the planning of long-term studies of vascular disease, ion transport experiments, and interpretation of the data. Future directions for our contributors' research will advance the field.

Specificity of Ca2+-activated K+ channel modulation in atherosclerosis and aerobic exercise training.

Vascular smooth muscle cells express several isoforms of a number of classes of K+ channels. Potassium channels play critical roles in the regulation of vascular smooth muscle contraction as well as vascular smooth muscle cell proliferation or phenotypic modulation. There is ample evidence that it is Ca2+ that enables these two seemingly disparate functions to be tightly coupled both in healthy and disease processes. Because of the central position that potassium channels have in vasocontraction, vasorelaxation, membrane potential, and smooth muscle cell proliferation, these channels continue to possess the potential to serve as novel therapeutic targets in cardiovascular disease. While there are questions that remain regarding the complete interactions between K+ channels, vascular regulation, smooth muscle cell proliferation, and phenotypic modulation in physiological and pathophysiological conditions, a broad understanding of the contributions of each class of K+ channel to contractile and proliferative states of the vasculature has been reached. This brief review will discuss the current understanding of the role of K+ channels in vascular smooth muscle cells in health and disease using the porcine vascular smooth muscle cell model with particular attention to new scientific discoveries contributed by the authors regarding the effect of endurance exercise on the function of the K+ channels.

Multiphasic changes in smooth muscle Ca2+ transporters during the progression of coronary atherosclerosis.

Ischemic heart disease due to macrovascular atherosclerosis and microvascular dysfunction is the major cause of death worldwide and the unabated increase in metabolic syndrome is a major reason why this will continue. Intracellular free Ca2+ ([Ca2+]i) regulates a variety of cellular functions including contraction, proliferation, migration, and transcription. It follows that studies of vascular Ca2+ regulation in reductionist models and translational animal models are vital to understanding vascular health and disease. Swine with metabolic syndrome (MetS) develop the full range of coronary atherosclerosis from mild to severe disease. Intravascular imaging enables quantitative measurement of atherosclerosis in vivo, so viable coronary smooth muscle (CSM) cells can be dispersed from the arteries to enable Ca2+ transport studies in native cells. Transition of CSM from the contractile phenotype in the healthy swine to the proliferative phenotype in mild atherosclerosis was associated with increases in SERCA activity, sarcoplasmic reticulum Ca2+, and voltage-gated Ca2+ channel function. In vitro organ culture confirmed that SERCA activation induces CSM proliferation. Transition from the proliferative to a more osteogenic phenotype was associated with decreases in all three Ca2+ transporters. Overall, there was a biphasic change in Ca2+ transporters over the progression of atherosclerosis in the swine model and this was confirmed in CSM from failing explanted hearts of humans. A major determinant of endolysosome content in human CSM is the severity of atherosclerosis. In swine CSM endolysosome Ca2+ release occurred through the TPC2 channel. We propose a multiphasic change in Ca2+ transporters over the progression of coronary atherosclerosis.

Atherosclerosis Burdens in Diabetes Mellitus: Assessment by PET Imaging.

Arteriosclerosis and its sequelae are the most common cause of death in diabetic patients and one of the reasons why diabetes has entered the top 10 causes of death worldwide, fatalities having doubled since 2000. The literature in the field claims almost unanimously that arteriosclerosis is more frequent or develops more rapidly in diabetic than non-diabetic subjects, and that the disease is caused by arterial inflammation, the control of which should therefore be the goal of therapeutic efforts. These views are mostly based on indirect methodologies, including studies of artery wall thickness or stiffness, or on conventional CT-based imaging used to demonstrate tissue changes occurring late in the disease process. In contrast, imaging with positron emission tomography and computed tomography (PET/CT) applying the tracers 18F-fluorodeoxyglucose (FDG) or 18F-sodium fluoride (NaF) mirrors arterial wall inflammation and microcalcification, respectively, early in the course of the disease, potentially enabling in vivo insight into molecular processes. The present review provides an overview of the literature from the more than 20 and 10 years, respectively, that these two tracers have been used for the study of atherosclerosis, with emphasis on what new information they have provided in relation to diabetes and which questions remain insufficiently elucidated.

Swine Disease Models for Optimal Vascular Engineering.

Swine disease models are essential for mimicry of human metabolic and vascular pathophysiology, thereby enabling high-fidelity translation to human medicine. The worldwide epidemic of obesity, metabolic disease, and diabetes has prompted the focus on these diseases in this review. We highlight the remarkable similarity between Ossabaw miniature swine and humans with metabolic syndrome and atherosclerosis. Although the evidence is strongest for swine models of coronary artery disease, findings are generally applicable to any vascular bed. We discuss the major strengths and weaknesses of swine models. The development of vascular imaging is an example of optimal vascular engineering in swine. Although challenges regarding infrastructure and training of engineers in the use of swine models exist, opportunities are ripe for gene editing, studies of molecular mechanisms, and use of swine in coronary artery imaging and testing of devices that can move quickly to human clinical studies.

Guidelines for animal exercise and training protocols for cardiovascular studies.

Whole body exercise tolerance is the consummate example of integrative physiological function among the metabolic, neuromuscular, cardiovascular, and respiratory systems. Depending on the animal selected, the energetic demands and flux through the oxygen transport system can increase two orders of magnitude from rest to maximal exercise. Thus, animal models in health and disease present the scientist with flexible, powerful, and, in some instances, purpose-built tools to explore the mechanistic bases for physiological function and help unveil the causes for pathological or age-related exercise intolerance. Elegant experimental designs and analyses of kinetic parameters and steady-state responses permit acute and chronic exercise paradigms to identify therapeutic targets for drug development in disease and also present the opportunity to test the efficacy of pharmacological and behavioral countermeasures during aging, for example. However, for this promise to be fully realized, the correct or optimal animal model must be selected in conjunction with reproducible tests of physiological function (e.g., exercise capacity and maximal oxygen uptake) that can be compared equitably across laboratories, clinics, and other proving grounds. Rigorously controlled animal exercise and training studies constitute the foundation of translational research. This review presents the most commonly selected animal models with guidelines for their use and obtaining reproducible results and, crucially, translates state-of-the-art techniques and procedures developed on humans to those animal models.

Atherosclerosis imaging with 18F-sodium fluoride PET: state-of-the-art review.

PURPOSE: We examined the literature to elucidate the role of 18F-sodium fluoride (NaF)-PET in atherosclerosis. METHODS: Following a systematic search of PubMed/MEDLINE, Embase, and Cochrane Library included articles underwent subjective quality assessment with categories low, medium, and high. Of 2811 records, 1780 remained after removal of duplicates. Screening by title and abstract left 41 potentially eligible full-text articles, of which 8 (about the aortic valve (n = 1), PET/MRI feasibility (n = 1), aortic aneurysms (n = 1), or quantification methodology (n = 5)) were dismissed, leaving 33 published 2010-2012 (n = 6), 2013-2015 (n = 11), and 2016-2018 (n = 16) for analysis. RESULTS: They focused on coronary (n = 8), carotid (n = 7), and femoral arteries (n = 1), thoracic aorta (n = 1), and infrarenal aorta (n = 1). The remaining 15 studies examined more than one arterial segment. The literature was heterogeneous: few studies were designed to investigate atherosclerosis, 13 were retrospective, 9 applied both FDG and NaF as tracers, 24 NaF only. Subjective quality was low in one, medium in 13, and high in 19 studies. The literature indicates that NaF is a very specific tracer that mimics active arterial wall microcalcification, which is positively associated with cardiovascular risk. Arterial NaF uptake often presents before CT-calcification, tends to decrease with increasing density of CT-calcification, and appears, rather than FDG-avid foci, to progress to CT-calcification. It is mainly surface localized, increases with age with a wide scatter but without an obvious sex difference. NaF-avid microcalcification can occur in fatty streaks, but the degree of progression to CT-calcification is unknown. It remains unknown whether medical therapy influences microcalcification. The literature held no therapeutic or randomized controlled trials. CONCLUSION: The literature was heterogeneous and with few clear cut messages. NaF-PET is a new approach to detect and quantify microcalcification in early-stage atherosclerosis. NaF uptake correlates with cardiovascular risk factors and appears to be a good measure of the body's atherosclerotic burden, potentially suited also for assessment of anti-atherosclerotic therapy.

Alloxan-induced diabetes exacerbates coronary atherosclerosis and calcification in Ossabaw miniature swine with metabolic syndrome.

BACKGROUND: There is a preponderance of evidence implicating diabetes with increased coronary artery disease (CAD) and calcification (CAC) in human patients with metabolic syndrome (MetS), but the effect of diabetes on CAD severity in animal models remains controversial. We investigated whether diabetes exacerbates CAD/CAC and intracellular free calcium ([Ca2+]i) dysregulation in the clinically relevant Ossabaw miniature swine model of MetS. METHODS: Sixteen swine, eight with alloxan-induced diabetes, were fed a hypercaloric, atherogenic diet for 6 months. Alloxan-induced pancreatic beta cell damage was examined by immunohistochemical staining of insulin. The metabolic profile was confirmed by body weight, complete blood panel, intravenous glucose tolerance test (IVGTT), and meal tolerance test. CAD severity was assessed with intravascular ultrasound and histology. [Ca2+]i handling in coronary smooth muscle (CSM) cells was assessed with fura-2 ratiometric imaging. RESULTS: Fasting and post-prandial blood glucose, total cholesterol, and serum triglycerides were elevated in MetS-diabetic swine. This group also exhibited hypoinsulinemia during IVGTT and less pancreatic beta cell mass when compared to lean and MetS-nondiabetic swine. IVUS analysis revealed that MetS-diabetic swine had greater percent wall coverage, percent plaque burden, and calcium index when compared to lean and MetS-nondiabetic swine. Fura-2 imaging of CSM [Ca2+]i revealed that MetS-nondiabetic swine exhibited increased sarcoplasmic reticulum Ca2+ store release and Ca2+ influx through voltage-gated Ca2+ channels compared to lean swine. MetS-diabetic swine exhibited impaired Ca2+ efflux. CONCLUSIONS: Diabetes exacerbates coronary atherosclerosis and calcification in Ossabaw miniature swine with MetS, accompanied by progression of [Ca2+]i dysregulation in advanced CAD/CAC. These results recapitulate increased CAD in humans with diabetes and establish Ossabaw miniature swine as an animal model for future MetS/diabetes comorbidity studies.

18F-NaF and 18F-FDG as molecular probes in the evaluation of atherosclerosis.

The early detection of atherosclerotic disease is vital to the effective prevention and management of life-threatening cardiovascular events such as myocardial infarctions and cerebrovascular accidents. Given the potential for positron emission tomography (PET) to visualize atherosclerosis earlier in the disease process than anatomic imaging modalities such as computed tomography (CT), this application of PET imaging has been the focus of intense scientific inquiry. Although 18F-FDG has historically been the most widely studied PET radiotracer in this domain, there is a growing body of evidence that 18F-NaF holds significant diagnostic and prognostic value as well. In this article, we review the existing literature on the application of 18F-FDG and 18F-NaF as PET probes in atherosclerosis and present the findings of original animal and human studies that have examined how well 18F-NaF uptake correlates with vascular calcification and cardiovascular risk.

Long-term spironolactone treatment reduces coronary TRPC expression, vasoconstriction, and atherosclerosis in metabolic syndrome pigs.

Coronary transient receptor potential canonical (TRPC) channel expression is elevated in metabolic syndrome (MetS). However, differential contribution of TRPCs to coronary pathology in MetS is not fully elucidated. We investigated the roles of TRPC1 and TRPC6 isoforms in coronary arteries of MetS pigs and determined whether long-term treatment with a mineralocorticoid receptor inhibitor, spironolactone, attenuates coronary TRPC expression and associated dysfunctions. MetS coronary arteries exhibited significant atherosclerosis, endothelial dysfunction, and increased histamine-induced contractions. Immunohistochemical studies revealed that TRPC6 immunostaining was significantly greater in the medial layer of MetS pig coronary arteries compared to that in Lean pigs, whereas little TRPC6 immunostaining was found in atheromas. Conversely, TRPC1 immunostaining was weak in the medial layer but strong in MetS atheromas, where it was predominantly localized to macrophages. Spironolactone treatment significantly decreased coronary TRPC expression and dysfunctions in MetS pigs. In vivo targeted delivery of the dominant-negative (DN)-TRPC6 cDNA to the coronary wall reduced histamine-induced calcium transients in the MetS coronary artery medial layer, implying a role for TRPC6 in mediating calcium influx in MetS coronary smooth muscles. Monocyte adhesion was increased in Lean pig coronary arteries cultured in the presence of aldosterone; and spironolactone antagonized this effect, suggesting that coronary mineralocorticoid receptor activation may regulate macrophage infiltration. TRPC1 expression in atheroma macrophages was associated with advanced atherosclerosis, whereas medial TRPC6 upregulation correlated with increased histamine-induced calcium transients and coronary contractility. We propose that long-term spironolactone treatment may be a therapeutic strategy to decrease TRPC expression and coronary pathology associated with MetS.

Intracellular calcium increases in vascular smooth muscle cells with progression of chronic kidney disease in a rat model.

Background: Vascular smooth muscle cells (VSMCs) exhibit phenotypic plasticity, promoting vascular calcification and increasing cardiovascular risk. Changes in VSMC intracellular calcium ([Ca 2+ ] i ) are a major determinant of plasticity, but little is known about changes in [Ca 2+ ] i in chronic kidney disease (CKD). We have previously demonstrated such plasticity in aortas from our rat model of CKD and therefore sought to examine changes in [Ca 2+ ] i during CKD progression. Materials and Methods: We examined freshly isolated VSMCs from aortas of normal rats, Cy/+ rats (CKD) with early and advanced CKD, and advanced CKD rats treated without and with 3% calcium gluconate (CKD + Ca 2+ ) to lower parathyroid hormone (PTH) levels. [Ca 2+ ] i was measured with fura-2. Results: Cy/+ rats developed progressive CKD, as assessed by plasma levels of blood urea nitrogen, calcium, phosphorus, parathyroid hormone and fibroblast growth factor 23. VSMCs isolated from rats with CKD demonstrated biphasic alterations in resting [Ca 2+ ] i : VSMCs from rats with early CKD exhibited reduced resting [Ca 2+ ] i , while VSMCs from rats with advanced CKD exhibited elevated resting [Ca 2+ ] i . Caffeine-induced sarcoplasmic reticulum (SR) Ca 2+ store release was modestly increased in early CKD and was more drastically increased in advanced CKD. The advanced CKD elevation in SR Ca 2+ store release was associated with a significant increase in the activity of the sarco-endoplasmic reticulum Ca 2+ ATPase (SERCA); however, SERCA2a protein expression was decreased in advanced CKD. Following SR Ca 2+ store release, recovery of [Ca 2+ ] i in the presence of caffeine and extracellular Ca 2+ was attenuated in VSMCs from rats with advanced CKD. This impairment, together with reductions in expression of the Na + /Ca 2+ exchanger, suggest a reduction in Ca 2+ extrusion capability. Finally, store-operated Ca 2+ entry (SOCE) was assessed following SR Ca 2+ store depletion. Ca 2+ entry during recovery from caffeine-induced SR Ca 2+ store release was elevated in advanced CKD, suggesting a role for exacerbated SOCE with progressing CKD. Conclusions: With progressive CKD in the Cy/+ rat there is increased resting [Ca 2+ ] i in VSMCs due, in part, to increased SOCE and impaired calcium extrusion from the cell. Such changes may predispose VSMCs to phenotypic changes that are a prerequisite to calcification.

Effect of renal shock wave lithotripsy on the development of metabolic syndrome in a juvenile swine model: a pilot study.

PURPOSE: We performed a pilot study to assess whether renal shock wave lithotripsy influences metabolic syndrome onset and severity. MATERIALS AND METHODS: Three-month-old juvenile female Ossabaw miniature pigs were treated with shock wave lithotripsy (2,000 shock waves at 24 kV with 120 shock waves per minute in 2) or sham shock wave lithotripsy (no shock waves in 2). Shock waves were targeted to the upper pole of the left kidney to model treatment that would also expose the pancreatic tail to shock waves. Pigs were then instrumented to directly measure arterial blood pressure via an implanted radiotelemetry device. They later received a hypercaloric atherogenic diet for about 7 months. Metabolic syndrome development was assessed by the intravenous glucose tolerance test. RESULTS: Metabolic syndrome progression and severity were similar in the sham treated and lithotripsy groups. The only exception arterial blood pressure, which remained relatively constant in sham treated pigs but began to increase at about 2 months towards hypertensive levels in lithotripsy treated pigs. Metabolic data on the 2 groups were pooled to provide a more complete assessment of metabolic syndrome development and progression in this juvenile pig model. The intravenous glucose tolerance test revealed substantial insulin resistance with impaired glucose tolerance within 2 months on the hypercaloric atherogenic diet with signs of further metabolic impairment at 7 months. CONCLUSIONS: These preliminary results suggest that renal shock wave lithotripsy is not a risk factor for worsening glucose tolerance or diabetes mellitus onset. However, it appears to be a risk factor for early onset hypertension in metabolic syndrome.

Perivascular adipose tissue potentiates contraction of coronary vascular smooth muscle: influence of obesity.

BACKGROUND: This investigation examined the mechanisms by which coronary perivascular adipose tissue (PVAT)-derived factors influence vasomotor tone and the PVAT proteome in lean versus obese swine. METHODS AND RESULTS: Coronary arteries from Ossabaw swine were isolated for isometric tension studies. We found that coronary (P=0.03) and mesenteric (P=0.04) but not subcutaneous adipose tissue augmented coronary contractions to KCl (20 mmol/L). Inhibition of CaV1.2 channels with nifedipine (0.1 µmol/L) or diltiazem (10 µmol/L) abolished this effect. Coronary PVAT increased baseline tension and potentiated constriction of isolated arteries to prostaglandin F2α in proportion to the amount of PVAT present (0.1-1.0 g). These effects were elevated in tissues obtained from obese swine and were observed in intact and endothelium denuded arteries. Coronary PVAT also diminished H2O2-mediated vasodilation in lean and, to a lesser extent, in obese arteries. These effects were associated with alterations in the obese coronary PVAT proteome (detected 186 alterations) and elevated voltage-dependent increases in intracellular [Ca(2+)] in obese smooth muscle cells. Further studies revealed that the Rho-kinase inhibitor fasudil (1 µmol/L) significantly blunted artery contractions to KCl and PVAT in lean but not obese swine. Calpastatin (10 µmol/L) also augmented contractions to levels similar to that observed in the presence of PVAT. CONCLUSIONS: Vascular effects of PVAT vary according to anatomic location and are influenced by an obese phenotype. Augmented contractile effects of obese coronary PVAT are related to alterations in the PVAT proteome (eg, calpastatin), Rho-dependent signaling, and the functional contribution of K(+) and CaV1.2 channels to smooth muscle tone.

Shock wave lithotripsy targeting of the kidney and pancreas does not increase the severity of metabolic syndrome in a porcine model.

PURPOSE: We determined whether shock wave lithotripsy of the kidney of pigs with metabolic syndrome would worsen glucose tolerance or increase the risk of diabetes mellitus. MATERIALS AND METHODS: Nine-month-old female Ossabaw miniature pigs were fed a hypercaloric atherogenic diet to induce metabolic syndrome. At age 15 months the pigs were treated with 2,000 or 4,000 shock waves (24 kV at 120 shock waves per minute) using an unmodified HM3 lithotripter (Dornier MedTech, Kennesaw, Georgia). Shock waves were targeted to the left kidney upper pole calyx to model treatment that would also expose the pancreatic tail to shock waves. The intravenous glucose tolerance test was done in conscious fasting pigs before lithotripsy, and 1 and 2 months after lithotripsy with blood samples taken for glucose and insulin measurement. RESULTS: Pigs fed the hypercaloric atherogenic diet were obese, dyslipidemic, insulin resistant and glucose intolerant, consistent with metabolic syndrome. Assessments of insulin resistance, glucose tolerance and pancreatic ß cell function from fasting plasma glucose and insulin levels, and the glucose and insulin response profile to the intravenous glucose tolerance test were similar before and after lithotripsy. CONCLUSIONS: The metabolic syndrome status of pigs treated with shock wave lithotripsy was unchanged 2 months after kidney treatment with 2,000 high amplitude shock waves or overtreatment with 4,000 high amplitude shock waves. These findings do not support a single shock wave lithotripsy treatment of the kidney as a risk factor for the onset of diabetes mellitus.

Insulin-Dendrimer Nanocomplex for Multi-Day Glucose-Responsive Therapy in Mice and Swine.

The management of diabetes in a manner offering autonomous insulin therapy responsive to glucose-directed need, and moreover with a dosing schedule amenable to facile administration, remains an ongoing goal to improve the standard of care. While basal insulins with reduced dosing frequency, even once-weekly administration, are on the horizon, there is still no approved therapy that offers glucose-responsive insulin function. Herein, a nanoscale complex combining both electrostatic- and dynamic-covalent interactions between a synthetic dendrimer carrier and an insulin analogue modified with a high-affinity glucose-binding motif yields an injectable insulin depot affording both glucose-directed and long-lasting insulin availability. Following a single injection, it is even possible to control blood glucose for at least one week in diabetic swine subjected to daily oral glucose challenges. Measurements of serum insulin concentration in response to challenge show increases in insulin corresponding to elevated blood glucose levels, an uncommon finding even in preclinical work on glucose-responsive insulin. Accordingly, the subcutaneous nanocomplex that results from combining electrostatic- and dynamic-covalent interactions between a modified insulin and a synthetic dendrimer carrier affords a glucose-responsive insulin depot for week-long control following a single routine injection.

Flipped classroom model improves graduate student performance in cardiovascular, respiratory, and renal physiology.

The purpose of this study was to assess the effectiveness of a traditional lecture-based curriculum versus a modified "flipped classroom" curriculum of cardiovascular, respiratory, and renal physiology delivered to first-year graduate students. Students in both courses were provided the same notes and recorded lectures. Students in the modified flipped classroom were required to watch the prerecorded lectures before class and then attend class, where they received a quiz or homework covering material in each lecture (valued at 25% of the final grade) followed by a question and answer/problem-solving period. In the traditional curriculum, attending lectures was optional and there were no quizzes. Evaluation of effectiveness and student performance was achieved by having students in both courses take the same multiple-choice exams. Within a comparable group of graduate students, participants in the flipped course scored significantly higher (P ≤ 0.05) on the cardiovascular, respiratory, and weighted cumulative sections by an average of >12 percentage points. Exam averages for students in the flipped course also tended to be higher on the renal section by ∼11 percentage points (P = 0.06). Based on our experience and responses obtained in blinded student surveys, we propose that the use of homework and in-class quizzes were critical motivating factors that likely contributed to the increase in student exam performance. Taken together, our findings support that the flipped classroom model is a highly effective means in which to disseminate key physiological concepts to graduate students.

Label-free quantitative imaging of cholesterol in intact tissues by hyperspectral stimulated Raman scattering microscopy.

A finger on the pulse: Current molecular analysis of cells and tissues routinely relies on separation, enrichment, and subsequent measurements by various assays. Now, a platform of hyperspectral stimulated Raman scattering microscopy has been developed for the fast, quantitative, and label-free imaging of biomolecules in intact tissues using spectroscopic fingerprints as the contrast mechanism.