Comparison of Biomarkers of Oxidative Stress and Cardiovascular Disease in Humans and Chimpanzees (Pan troglodytes)

In the oxidative stress hypothesis of aging, the aging process is the result of cumulative damage by reactive oxygen species. Humans and chimpanzees are remarkably similar; but humans live twice as long as chimpanzees and therefore are believed to age at a slower rate. The purpose of this study was to compare biomarkers for cardiovascular disease, oxidative stress, and aging between male chimpanzees and humans. Compared with men, male chimpanzees were at increased risk for cardiovascular disease because of their significantly higher levels of fibrinogen, IGF1, insulin, lipoprotein a, and large high-density lipoproteins. Chimpanzees showed increased oxidative stress, measured as significantly higher levels of 5-hydroxymethyl-2-deoxyuridine and 8-iso-prostaglandin F_2α, a higher peroxidizability index, and higher levels of the prooxidants ceruloplasmin and copper. In addition, chimpanzees had decreased levels of antioxidants, including α- and β-carotene, β-cryptoxanthin, lycopene, and tocopherols, as well as decreased levels of the cardiovascular protection factors albumin and bilirubin. As predicted by the oxidative stress hypothesis of aging, male chimpanzees exhibit higher levels of oxidative stress and a much higher risk for cardiovascular disease, particularly cardiomyopathy, compared with men of equivalent age. Given these results, we hypothesize that the longer lifespan of humans is at least in part the result of greater antioxidant capacity and lower risk of cardiovascular disease associated with lower oxidative stress.Abbreviations: 5OHmU, 5-hydroxymethyl-2-deoxyuridine; 8isoPGF_2α, 8-iso-prostaglandin F_2α; HDL, high-density lipoprotein; IGF1, insulin-like growth factor 1; LDL, low-density lipoprotein; ROS, reactive oxygen speciesAging is characterized as a progressive reduction in the capacity to withstand the stresses of everyday life and a corresponding increase in risk of mortality. According to the oxidative stress hypothesis of aging, much of the aging process can be accounted for as the result of cumulative damage produced by reactive oxygen species (ROS).^{6,21,28,41,97} Endogenous oxygen radicals (that is, ROS) are generated as a byproduct of normal metabolic reactions in the body and subsequently can cause extensive damage to proteins, lipids, and DNA.^6,41 Various prooxidant elements, in particular free transition metals, can catalyze these destructive reactions.⁶ The damage caused by ROS can be counteracted by antioxidant defense systems, but the imbalance between production of ROS and antioxidant defenses, over time, leads to oxidative stress and may contribute to the rate of aging.^28,97Oxidative stress has been linked to several age-related diseases including neurodegenerative diseases, ophthalmologic diseases, cancer, and cardiovascular disease.^21,28,97 Of these, cardiovascular disease remains the leading cause of adult death in the United States and Europe.⁷¹ In terms of cardiovascular disease, oxidative stress has been linked to atherosclerosis, hypertension, cardiomyopathy, and chronic heart failure in humans.^55,78,84 Increases in oxidant catalysts (prooxidants)—such as copper, iron, and cadmium—have been associated with hypertension, coronary artery disease, atherosclerosis, and sudden cardiac death.^98,102,106 Finally, both endogenous and exogenous antioxidants have been linked to decreased risk of cardiovascular disease, although the mechanisms behind this relationship are unclear.^11,52,53 However, the oxidative stress hypothesis of aging aims to explain not only the mechanism of aging and age-related diseases (such as cardiovascular disease) in humans but also the differences between aging rates and the manifestations of age-related diseases across species.The differences in antioxidant and ROS levels between animals and humans offer promise for increasing our understanding of human aging. Additional evidence supporting the oxidative stress hypothesis of aging has come from comparative studies linking differences in aging rates across taxa with both antioxidant and ROS levels.^{4,17-21,58,71,86,105} In mammals, maximum lifespan potential is positively correlated with both serum and tissue antioxidant levels.^{17,18,21,71,105} Research has consistently demonstrated that the rate of oxidative damage varies across species and is negatively correlated with maximum lifespan potential.^{4,19,20,58,71,86} However, few studies involved detailed comparisons of hypothesized biochemical indicators of aging and oxidative stress between humans and animals.⁶ This type of interspecies comparison has great potential for directly testing the oxidative stress hypothesis of aging.Much evolutionary and genetic evidence supports remarkable similarity between humans and chimpanzees.^95,100 Despite this similarity, humans have a lifespan of almost twice that of chimpanzees.^3,16,47 Most comparative primate aging research has focused on the use of a macaque model,^62,81,88 and several biochemical markers of age-related diseases have been identified in both humans and macaque monkeys.^{9,22,28,81,93,97} Several other species of monkeys have also been used in research addressing oxidative stress, antioxidant defenses, and maximum lifespan potential.^18,21,58,105 However, no study to date has examined biochemical indicators of oxidative stress and aging in chimpanzees and humans as a test of the oxidative stress hypothesis for aging. The purpose of this study is to compare biochemical markers for cardiovascular disease, oxidative stress, and aging directly between male chimpanzees and humans. Given the oxidative stress hypothesis for aging and the known role of oxidative stress in cardiovascular disease, we predict that chimpanzees will show higher levels of cardiovascular risk and oxidative stress than humans.     

Age- and Sex-Associated Effects on Acute-Phase Proteins in G?ttingen Minipigs

Göttingen minipigs are a useful model for diseases having an inflammatory component, and the associated use of acute-phase proteins (APP) as biomarkers of inflammation warrants establishment of their reference ranges. The objective of this study was to establish reference values for selected APP in Göttingen minipigs and to investigate the effects of age, sex, and various stimuli on these ranges. Serum concentrations of C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin, pig major acute-phase protein (PMAP), albumin, and porcine α-1 acid glycoprotein (PAGP) were evaluated in 4 age groups (6, 16, 24 and 40–48 wk) of male and female Göttingen minipigs. In addition, minipigs were tested under 2 housing conditions, after acute LPS challenge, and after diet-induced obesity with and without mild diabetes. Changing the pigs to a new environment induced significant increases in CRP, PMAP, haptoglobin and PAGP and a decrease in albumin. An acute LPS stimulus increased CRP, PMAP, haptoglobin, and SAA; PAGP was unchanged and albumin decreased. Obese pigs with and without diabetes showed increases in CRP and PAGP, albumin decreased, and haptoglobin and SAA were unchanged. PMAP was increased only in obese pigs without diabetes. In conclusion, reference values for CRP, PMAP, haptoglobin, SAA, PAGP and albumin were established for male and female Göttingen minipigs of different ages. These APP were influenced by age and sex, underlining the importance of considering these factors when designing and interpreting studies including aspects of inflammation. In addition, an APP response was verified after both acute and chronic stimuli. Abbreviations: APP, acute-phase proteins; APR, acute-phase response; CRP, C-reactive protein; HFD, high-fat diet; HFD+D, high fat diet + diabetes; PAGP, porcine α1 acid glycoprotein; PMAP, pig major acute-phase protein; SAA, serum amyloid AInflammation is involved in a number of important and increasingly widespread human diseases, including inflammatory bowel diseases, cancers, infections, metabolic diseases like obesity and diabetes, and cardiovascular diseases like atherosclerosis.^{1,5,7,11,20,41} The systemic response to inflammation is the acute-phase response (APR) which, together with innate immune responses, prevents infection, clears pathogens, and contributes to inflammation resolution and the healing process. The APR has been extensively described in humans^10,22 and other mammals,^8,14,29,31 and in all cases, it is regulated by cytokines including IL6 and TNFα.^21,30 The APR is activated by many different stimuli, including trauma, infection, stress, neoplasia, and inflammatory stimuli, resulting in significant changes in the circulating concentrations of the so-called acute-phase proteins (APP). The APP are synthetized primarily by the liver and can be divided into positive and negative APP depending on whether their concentration in plasma increases (positive) or decreases (negative) in response to a stimulus.¹⁰ In addition, they can be divided into major and minor APP, depending on the magnitude of their concentration change after a given stimulus.²² Because the concentrations of the APP change in response to a given stimulus, their serum or plasma levels can be used diagnostically as biomarkers of disease severity and progression or to evaluate the effect of various interventions.^8,14,31 The APP show different kinetics after a stimulus, with C-reactive protein (CRP) and serum amyloid A (SAA) displaying rapid increases and normalization after the stimulus has been removed, whereas haptoglobin shows a later and more prolonged response.^10,31 The APR may be transient and revert to normal with recovery, or it can persist, as during chronic conditions.²¹ Importantly, APP and their kinetics differ somewhat between species.³¹To further elucidate the involvement of inflammation in human diseases, accurate animal models of inflammation, including species validated biomarkers of inflammation, are needed. Mouse models are commonly used in many research areas, but their response to several different inflammatory conditions is not comparable to that of humans, and therefore the predictive validity of these models may be limited.³⁹ Pigs are highly comparable to humans with respect to anatomy and physiology,⁴⁴ and their APR to various stimuli has been described.^14,23,26 In general, the APR and the resulting changes in APP seem to be very similar in pigs compared with humans, with CRP, haptoglobin, and SAA being major positive APP and albumin being a negative APP.¹⁴ In humans, α1-acid glycoprotein (AGP) is a positive APP but has been reported to either increase,¹⁷ remain unchanged^23,45 or to decrease¹² in pigs, depending on the stimulus investigated. The concentrations of some of the major APP characterized in domestic pigs show significant effects of age and sex.^32,34 In addition to age and sex effects, significant differences in APP between herds have been observed, most likely reflecting different pathogenic pressures in the different herds.³² Furthermore, some indications exist for possible interbreed differences in APP concentrations, although this possibility has not been investigated in detail.¹²Minipigs are especially relevant in biomedical research, given their smaller size and well-defined microbiology and genetics.⁴ Göttingen minipigs are a useful model for several conditions involving inflammation and the APR, including infection,² obesity,¹⁹ diabetes²⁴ and atherosclerosis,¹⁸ and different APP have already been used as biomarkers in some of these models.² Therefore, existing data suggest that APP commonly applied in human medicine could be relevant in Göttingen minipigs as well. However, the APR and reference values of APP, including the potential influence of age and sex indicated in other studies, have not been investigated systematically in this breed.^12,32,34The objective of the current study was to establish reference values of selected APP in normal Göttingen minipigs, including evaluation of the possible effects of age and sex. In addition, the effects of housing condition and acute and chronic inflammatory stimuli were assessed.     

Anatomic,Hematologic, and Biochemical Features of C57BL/6NCrl Mice Maintained on Chronic Oral Corticosterone

Metabolic syndrome is a condition that typically includes central obesity, insulin resistance, glucose intolerance, dyslipidemia, and hypertension. Disruption of the hypothalamic–pituitary–adrenal axis, a regulator of corticosterone secretion, occurs in some cases of metabolic syndrome and obesity, and Cushing hypercortisolemia is associated with obesity and metabolic disorders. We therefore assessed anatomic and clinical pathology in C57BL/6NCrl mice to evaluate the effects of chronic corticosterone in the drinking water at doses of 25, 50, and 100 μg/mL for 25 d. Treated mice developed obesity, glucose intolerance, electrolyte aberrations, and dyslipidemia that were dose-dependent and most severe in the 100-μg/mL treatment group. To evaluate return to normal function, additional C57BL/6NCrl mice received corticosterone-free water for 2 wk after the 25-d treatment period. According to results of gross examination, mice appeared to recover within days of exogenous corticosterone withdrawal; however, adrenal gland vacuolation and protein, lipid, and electrolyte abnormalities persisted. Together, these findings support chronic corticosterone exposure through the drinking water as a potentially useful, noninvasive method to induce some features of metabolic syndrome.Obesity and associated metabolic dysfunctions are an increasing public health concern in modern Western society. In humans, obesity and metabolic syndrome heighten the risk of developing debilitating and costly illness including diabetes, cardiovascular disease, stroke, and some forms of cancer.^2,20 Mounting evidence indicates that stress and associated hormones such as cortisol (corticosterone in rodents) contribute to the development of metabolic syndrome. Furthermore, regional glucocorticoid metabolism in adipocytes is proposed to be involved in the pathogenesis of metabolic syndrome.^{6,16,17,27,56} Cushing syndrome, iatrogenic hypercortisolemia, and metabolic syndrome share clinical and physiologic similarities, including central obesity, insulin resistance, glucose intolerance, dyslipidemia, and hypertension.^{1,2,31,35,41,46} How glucocorticoids contribute to the development of these problems remains unclear.Numerous clinical and experimental studies have linked stress, diet, and lifestyle choices to changes in risk factors associated with the development of metabolic disorders.^{1,3,7,10,21,33,36,42,55} How corticosterone influences this risk remains unclear. Although corticosterone has beneficial short-term effects, long-term corticosterone exposure can result in damage to the physiologic systems it protects acutely.²⁷ Disruption of this physiologic signal occurs in numerous disparate disorders, ranging from depression to Cushing syndrome.^16,22,36,54 Therefore, understanding the effects of chronic high corticosterone on metabolism and physiology is of key importance.To clarify how chronic treatment with corticosterone alters the physiology of an organism, we treated adrenally intact adult male mice with corticosterone in drinking water for 4 wk. Furthermore, we examined the return of physiology 2 wk after withdrawal of chronic corticosterone administration. We used this approach as a rapid (3- to 4-wk), noninvasive method of altering plasma corticosterone levels that enabled us to retain some integrity in the diurnal rhythm present in normal animals.We previously characterized the gross metabolic consequences of exogenous noninvasive corticosterone delivery in the drinking water.^20,28 In those studies, we found that high doses of corticosterone (100 μg/mL) resulted in rapid and dramatic hyperphagia; weight gain; increased adiposity; elevated plasma corticosterone, leptin, insulin, and triglyceride levels; and decreased homecage locomotion.²⁰ Moreover, several studies have shown that a lower dose of corticosterone (25 μg/mL) resulted in an intermediate phenotype in some of these measures but had no effect on others.^{12,14,20,23,28,38,42,47} As such, the high corticosterone dose results in a phenotype that satisfies most of the criteria for metabolic syndrome as defined by the National Heart, Lung, and Blood Institute and the American Heart Association.¹⁵ However, little information is available on the resulting histologic, hematologic, and serum chemical profiles associated with this treatment. We sought to more fully characterize this model to support selection of the model that most accurately reflects the human disease conditions under study. In-depth characterization of the model also provides more precise measurements of response to therapies intended to ameliorate the effects of the treatment.The current study provides a detailed examination of the physiologic effect of 3 dosages of corticosterone—low (25 μg/mL), intermediate (50 μg/mL), and high (100 μg/mL) doses—in drinking water. The goal was to extend the previous findings that established this regimen as a model of metabolic syndrome by exploring the detailed physiologic changes associated with this model and to assess whether and how treated mice recover after withdrawal of the corticosterone treatment. We propose that the physiologic changes observed in the mice treated with high-dose corticosterone approximate changes observed in human patients with metabolic syndrome and that these mice potentially serve as a model for hypercortisolemia and associated obesity. In addition, we hypothesized that 2 wk of recovery from corticosterone treatment would not completely resolve cellular and clinical pathologies characterized during treatment, given the numerous changes in physiology.     

Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

Multivesicular bodies (MVBs) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these ILV contain Fas ligand (FasL) and are secreted as ''lethal exosomes'' following activation-induced fusion of the MVB with the plasma membrane. Diacylglycerol (DAG) and diacylglycerol kinase α (DGKα) regulate MVB maturation and polarized traffic, as well as subsequent secretion of pro-apoptotic exosomes, but the molecular basis underlying these phenomena remains unclear. Here we identify protein kinase D (PKD) family members as DAG effectors involved in MVB genesis and secretion. We show that the inducible secretion of exosomes is enhanced when a constitutively active PKD1 mutant is expressed in T lymphocytes, whereas exosome secretion is impaired in PKD2-deficient mouse T lymphoblasts and in PKD1/3-null B cells. Analysis of PKD2-deficient T lymphoblasts showed the presence of large, immature MVB-like vesicles and demonstrated defects in cytotoxic activity and in activation-induced cell death. Using pharmacological and genetic tools, we show that DGKα regulates PKD1/2 subcellular localization and activation. Our studies demonstrate that PKD1/2 is a key regulator of MVB maturation and exosome secretion, and constitutes a mediator of the DGKα effect on MVB secretory traffic.Exosomes are nanovesicles that form as intraluminal vesicles (ILVs) inside multivesicular bodies (MVBs) and are then secreted by numerous cell types.¹ ILVs are generated by inward budding of late endosome limiting membrane in a precisely regulated maturation process.^{2, 3} Two main pathways are involved in MVB maturation.^{4, 5} In addition to the ESCRT (endosomal complex required for traffic) proteins,⁶ there is increasing evidence that lipids such as lyso-bisphosphatidic acid (LBPA),⁷ ceramides⁸ and diacylglycerol (DAG)⁹ contribute to this membrane invagination process.Exosomes participate in many biological processes related to T-cell receptor (TCR)-triggered immune responses, including T lymphocyte-mediated cytotoxicity and activation-induced cell death (AICD), antigen presentation and intercellular miRNA exchange.^{10, 11, 12, 13, 14, 15} The discovery of exosome involvement in these responses increased interest in the regulation of exosome biogenesis and secretory traffic, with special attention to the contribution of lipids such as ceramide and DAG, as well as DAG-binding proteins.^{14, 16, 17, 18, 19, 20, 21} These studies suggest that positive and negative DAG regulators may control secretory traffic. By transforming DAG into phosphatidic acid (PA), diacylglycerol kinase α (DGKα) is essential for the negative control of DAG function in T lymphocytes.²² DGKα translocates transiently to the T-cell membrane after human muscarinic type 1 receptor (HM1R) triggering or to the immune synapse (IS) after TCR stimulation; at these subcellular locations, DGKα acts as a negative modulator of phospholipase C (PLC)-generated DAG.^{23, 24}The secretory vesicle pathway involves several DAG-controlled checkpoints at which DGKα may act; these include vesicle formation and fission at the trans-Golgi network (TGN), MVB maturation, as well as their transport, docking and fusion to the plasma membrane.^{9, 16, 17, 18, 19, 20} The molecular components that regulate some of these trafficking processes include protein kinase D (PKD) family members.²¹ PKD1 activity, for instance, regulates fission of transport vesicles from TGN via direct interaction with the pre-existing DAG pool at this site.¹⁹ The cytosolic serine/threonine kinases PKD1, PKD2 and PKD3^{(ref. 21)} are expressed in a wide range of cells, with PKD2 the most abundant isotype in T lymphocytes.^{25, 26} PKD have two DAG-binding domains (C1a and C1b) at the N terminus,²¹ which mediate PKD recruitment to cell membranes. Protein kinase C (PKC) phosphorylation at the PKD activation loop further promotes PKD autophosphorylation and activation.²⁷Based on our previous studies showing DGKα regulation of DAG in MVB formation and exosome secretion,^{9, 14, 28} and the identification of PKD1/2 association to MVB,¹⁴ we hypothesized that DGKα control of DAG mediates these events, at least in part, through PKD. Here we explored whether, in addition to its role in vesicle fission from TGN,¹⁹ PKD regulates other steps in the DAG-controlled secretory traffic pathway. Using PKD-deficient cell models, we analyzed the role of PKD1/2 in MVB formation and function, and demonstrate their implication in exosome secretory traffic.     

TRAF2 is a biologically important necroptosis suppressor

Tumor necrosis factor α (TNFα) triggers necroptotic cell death through an intracellular signaling complex containing receptor-interacting protein kinase (RIPK) 1 and RIPK3, called the necrosome. RIPK1 phosphorylates RIPK3, which phosphorylates the pseudokinase mixed lineage kinase-domain-like (MLKL)—driving its oligomerization and membrane-disrupting necroptotic activity. Here, we show that TNF receptor-associated factor 2 (TRAF2)—previously implicated in apoptosis suppression—also inhibits necroptotic signaling by TNFα. TRAF2 disruption in mouse fibroblasts augmented TNFα–driven necrosome formation and RIPK3-MLKL association, promoting necroptosis. TRAF2 constitutively associated with MLKL, whereas TNFα reversed this via cylindromatosis-dependent TRAF2 deubiquitination. Ectopic interaction of TRAF2 and MLKL required the C-terminal portion but not the N-terminal, RING, or CIM region of TRAF2. Induced TRAF2 knockout (KO) in adult mice caused rapid lethality, in conjunction with increased hepatic necrosome assembly. By contrast, TRAF2 KO on a RIPK3 KO background caused delayed mortality, in concert with elevated intestinal caspase-8 protein and activity. Combined injection of TNFR1-Fc, Fas-Fc and DR5-Fc decoys prevented death upon TRAF2 KO. However, Fas-Fc and DR5-Fc were ineffective, whereas TNFR1-Fc and interferon α receptor (IFNAR1)-Fc were partially protective against lethality upon combined TRAF2 and RIPK3 KO. These results identify TRAF2 as an important biological suppressor of necroptosis in vitro and in vivo.Apoptotic cell death is mediated by caspases and has distinct morphological features, including membrane blebbing, cell shrinkage and nuclear fragmentation.^{1, 2, 3, 4} In contrast, necroptotic cell death is caspase-independent and is characterized by loss of membrane integrity, cell swelling and implosion.^{1, 2, 5} Nevertheless, necroptosis is a highly regulated process, requiring activation of RIPK1 and RIPK3, which form the core necrosome complex.^{1, 2, 5} Necrosome assembly can be induced via specific death receptors or toll-like receptors, among other modules.^{6, 7, 8, 9} The activated necrosome engages MLKL by RIPK3-mediated phosphorylation.^{6, 10, 11} MLKL then oligomerizes and binds to membrane phospholipids, forming pores that cause necroptotic cell death.^{10, 12, 13, 14, 15} Unchecked necroptosis disrupts embryonic development in mice and contributes to several human diseases.^{7, 8, 16, 17, 18, 19, 20, 21, 22}The apoptotic mediators FADD, caspase-8 and cFLIP suppress necroptosis.^{19, 20, 21, 23, 24} Elimination of any of these genes in mice causes embryonic lethality, subverted by additional deletion of RIPK3 or MLKL.^{19, 20, 21, 25} Necroptosis is also regulated at the level of RIPK1. Whereas TNFα engagement of TNFR1 leads to K63-linked ubiquitination of RIPK1 by cellular inhibitor of apoptosis proteins (cIAPs) to promote nuclear factor (NF)-κB activation,²⁶ necroptosis requires suppression or reversal of this modification to allow RIPK1 autophosphorylation and consequent RIPK3 activation.^{2, 23, 27, 28} CYLD promotes necroptotic signaling by deubiquitinating RIPK1, augmenting its interaction with RIPK3.²⁹ Conversely, caspase-8-mediated CYLD cleavage inhibits necroptosis.²⁴TRAF2 recruits cIAPs to the TNFα-TNFR1 signaling complex, facilitating NF-κB activation.^{30, 31, 32, 33} TRAF2 also supports K48-linked ubiquitination and proteasomal degradation of death-receptor-activated caspase-8, curbing apoptosis.³⁴ TRAF2 KO mice display embryonic lethality; some survive through birth but have severe developmental and immune deficiencies and die prematurely.^{35, 36} Conditional TRAF2 KO leads to rapid intestinal inflammation and mortality.³⁷ Furthermore, hepatic TRAF2 depletion augments apoptosis activation via Fas/CD95.³⁴ TRAF2 attenuates necroptosis induction in vitro by the death ligands Apo2L/TRAIL and Fas/CD95L.³⁸ However, it remains unclear whether TRAF2 regulates TNFα-induced necroptosis—and if so—how. Our present findings reveal that TRAF2 inhibits TNFα necroptotic signaling. Furthermore, our results establish TRAF2 as a biologically important necroptosis suppressor in vitro and in vivo and provide initial insight into the mechanisms underlying this function.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

Cerebral Amyloid Angiopathy in an Aged Sooty Mangabey (Cercocebus atys)

A 26-y-old male sooty mangabey (Cercocebus atys) was found at necropsy to have a moderate degree of cerebral amyloid β (Aβ) angiopathy in superficial and parenchymal blood vessels of the brain. Senile (Aβ) plaques were absent, as were neurofibrillary tangles and other signs of neurodegeneration. Affected blood vessels were arterial, capillary, and, less frequently, venous in nature. Histologically, the Aβ40 isoform was more prevalent than was Aβ42. As in humans but unlike in squirrel monkeys, the density of lesions in this mangabey increased along a rostral-to-caudal gradient. Therefore mangabeys appear to conform to the general tendency of nonhuman primates by developing cerebral Aβ angiopathy in the absence of other indices of Alzheimer-type neuropathology.Abbreviations: Aβ, amyloid β, CAA, cerebral amyloid angiopathy, GFAP, glial fibrillary acidic protein, Iba 1, microglia-expressed calcium-binding proteinOne of the most common microvasculopathies in the aging human brain is cerebral amyloid angiopathy (CAA), a disorder in which various aggregation-prone proteins accumulate in the walls of parenchymal and meningeal blood vessels.^4,9 Most often, the amyloidogenic protein is amyloid β (Aβ), a cleavage product of the Aβ precursor protein and the essential component of senile plaques in Alzheimer disease.^13,43 In the brain vasculature, the basal lamina is a primary site of Aβ deposition.^25,35 Severely affected arterioles show a loss of smooth muscle cells in the tunica media, a weakening of the vascular wall and a propensity to rupture.^3,34 CAA thus increases the risk of intracerebral bleeding and may be responsible for as much as 20% of nontraumatic hemorrhagic stroke in elderly humans.^15,18,35 CAA is present to various degrees in virtually all cases of Alzheimer disease,^15,16,21 but it also occurs independently.²⁴ As is the case for other proteopathies, advancing age is a significant risk factor for CAA.^8,19In humans, CAA most often affects the arteries and arterioles of the brain, particularly those in the leptomeninges and cortex.^2,25 CAA is less frequent in veins and capillaries,²⁵ but capillary CAA can be prominent in some cases.^26,33 The occipital lobe is affected most often^1,32,37 but all cortical regions are vulnerable. CAA is variable in occurrence in the cerebellum and uncommon in deep telencephalic gray structures, white matter, and the brainstem,³⁶ except in severely affected cases.³²Although its specific role in the pathogenesis of Alzheimer disease remains uncertain, there is now strong evidence that dementia is exacerbated by CAA.¹⁴ Furthermore, CAA is independently linked to cognitive decline both in rare familial cases²⁰ and in older humans with idiopathic CAA.^2,20 Despite the prevalence of cerebrovascular amyloidosis in elderly humans, surprisingly little is known about its effect on the brain, in part because of a paucity of natural animal models that closely mimic the human disorder.^17,38Nonhuman primates offer a unique opportunity to view CAA from a comparative perspective, given that they normally generate human-sequence Aβ and develop severe cerebral Aβ amyloidosis in old age, generally in the absence of other changes that characterize Alzheimer disease.¹² Nonhuman primates have the additional advantage that, compared with humans, their relatively small brains enable exhaustive regional analysis of microscopic lesions, something that, for practical reasons, is seldom undertaken in the human brain. Here we present the first investigation of age-associated brain changes in sooty mangabeys, focusing in particular on Aβ deposition and related abnormalities. One of the 2 aged mangabeys analyzed had Aβ deposition in the brain which was almost exclusively in the form of CAA. Remarkably, the vessel types affected and the regional distribution of CAA more closely resembled the pattern seen in humans than that in other nonhuman primates, particularly squirrel monkeys.⁶ Differences and similarities in CAA among primate species could provide fresh insights into the development of cerebral amyloidosis and related disorders in older humans.     

Acute Portal Hypertension Models in Dogs: Low- and High-Flow Approaches

Effective animal models are needed to evaluate the feasibility of new techniques to assess portal hypertension (PH). Here we developed 2 canine models of acute PH by increasing intrasinusoidal resistance and by increasing the portal vein (PV) flow volume to test the efficacy of a noninvasive technique to evaluate PH. The acute low-flow PH model was based on embolization of liver circulation by using a gelatin sponge material. The acute high-flow PH model was based on increasing the PV flow volume by using an arteriovenous (A-V) shunt from the femoral artery and saline infusion. PV pressures and diameters were assessed before and after inducing PH. Pressure values and diameters were obtained from the inferior vena cava in 3 unmanipulated controls. The low-flow model of PH was repeatable and successfully increased PV pressure by an average of 16.5 mm Hg within 15 min. The high-flow model of PH failed to achieve increased PV pressures. However, saline supplementation of the portal circulation in the high-flow model led to mean increases in PV pressures of 12.8 mm Hg within 20 min. Pulsatility in the PV was decreased in the low-flow model and increased in the high-flow model relative to baseline. No changes in PV diameter were noted in either model. These acute PH models are relatively straightforward to implement and may facilitate the evaluation of new techniques to assess PH.Abbreviations: PH, portal hypertension; PV, portal vein; A-V shunt, arterial-venous shunt; IVC, inferior vena cavaPortal hypertension (PH) marks the silent progression of cirrhotic liver from a preclinical to a clinical phase, when symptoms associated with common complications like ascites, variceal bleeding, and encephalopathy manifest and can be fatal (mortality rates of 20% to 70%).^7,12,31,34 The hepatic venous pressure gradient is used clinically to diagnose PH, but the technique is invasive and is not routinely used for screening potential PH cases.^26,39 Development of a noninvasive technique to identify nascent PH has great clinical value.³⁹ The long-term goal of our project is to develop and evaluate a contrast-enhanced ultrasound imaging-based noninvasive pressure estimation technique^{9,10,17,20,37} as a means to identify PH. Before clinical testing of this approach, we conducted feasibility studies in animal models to confirm the accuracy of this new technique. Here we developed 2 canine acute PH models to test the efficacy of our noninvasive pressure estimation technique to evaluate PH.Current animal PH models include those in mice, rats, pigs, rabbits, and dogs.^{1,5,11,13,21,23,27,33,38,41} Guidelines for selecting a particular animal PH model (or for other hepatic abnormalities) have also been published.³⁰ In view of these guidelines, we selected dogs for our study, because the blood vessels of smaller animals may be difficult to catheterize and because some species, like swine, develop marked pulmonary hypertension during contrast-enhanced ultrasound imaging.³² Moreover, our team has extensive experience with canine experiments, and we have noted no side effects in dogs after the administration of the contrast material for ultrasound imaging.^9,10,17 Furthermore the physiology of dogs closely approximates that of humans.²³Animal models and clinical studies have shown that a pathophysiologic increase in intrahepatic vascular resistance ultimately leads to PH,^12,31 whereas etiologically the origin of PH may be pre-, intra-, or postsinusoidal.⁶ Overall, a modified form of Ohm''s law for fluid flow (∆P = Q × R, where ∆P is the change in pressure, Q is flow, and R is resistance) explains increases in portal vein (PV) pressures,¹ either by an increase in PV flow, an increase in resistance to PV flow, or a combination of these 2 mechanisms. Therefore, we sought to develop 2 acute PH models in dogs, with one resulting from increased resistance to PV flow (a low-flow model of PH) and the other from an increase in the volume of PV flow (a high-flow model of PH).For our acute low-flow model of PH, we increased the intrasinusoidal resistance in the liver parenchyma by injecting a gelatin sponge material (Gelfoam, Ethicon, Somerville, NJ) via the PV. This product has been used previously in dogs²⁸ and does not elicit foreign-body reactions or inflammation.²² We hypothesized that PV pressures would increase and the pulsatility of PV flow would decrease (due to the decrease in flow) after administration of the gelatin material. For the acute high-flow PH model, we used an arteriovenous (A-V) shunt, which was created by connecting the splenic or femoral artery to the PV. We hypothesized that, in this model, both PV pressure and pulsatility would increase (due to the superimposition of arterial flow) after initiating the high-flow conditions in the PV.     

Ret is essential to mediate GDNF's neuroprotective and neuroregenerative effect in a Parkinson disease mouse model

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival and regeneration-promoting factor for dopaminergic neurons in cell and animal models of Parkinson disease (PD). GDNF is currently tested in clinical trials on PD patients with so far inconclusive results. The receptor tyrosine kinase Ret is the canonical GDNF receptor, but several alternative GDNF receptors have been proposed, raising the question of which signaling receptor mediates here the beneficial GDNF effects. To address this question we overexpressed GDNF in the striatum of mice deficient for Ret in dopaminergic neurons and subsequently challenged these mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Strikingly, in this established PD mouse model, the absence of Ret completely abolished GDNF''s neuroprotective and regenerative effect on the midbrain dopaminergic system. This establishes Ret signaling as absolutely required for GDNF''s effects to prevent and compensate dopaminergic system degeneration and suggests Ret activation as the primary target of GDNF therapy in PD.Glial cell line-derived neurotrophic factor (GDNF) is the founding member of the four ligands in the GDNF family, which belong to the transforming growth factor-β superfamily.¹ GDNF was characterized as a potent survival factor for many neurons in culture such as dopaminergic, motor, sympathetic, parasympathetic, sensory and enteric neurons.^{1, 2} In addition, in dopaminergic neuron cultures GDNF stimulates neuronal differentiation, neurite outgrowth, synapse formation and dopamine release.^{1, 2}As degeneration of midbrain dopaminergic neurons in the substantia nigra pars compacta (SNpc) represents a major hallmark of Parkinson disease (PD), the most common neurodegenerative movement disorder, GDNF has raised considerable interest as a therapeutic molecule for the treatment of PD.^{3, 4, 5} PD affects >2% of individuals over the age of 60 years, but no curative treatment is available to date, mainly due to a lack of understanding disease etiology.^{6, 7, 8} Preclinical studies in the established 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) rodent and primate models of PD demonstrated a substantial neuroprotection and regeneration effect by striatal provided GDNF or its close relative neurturin.^{3, 4, 9} However, clinical phase II trials on PD patients using GDNF or neurturin did so far not convincingly recapitulate their beneficial effects on the dopaminergic system in humans most likely due to technical problems and the selection of advanced PD patients.^{10, 11, 12, 13}GDNF signaling is highly complex as this neurotrophic factor can bind to a variety of receptors, thus being able to induce pleiotropic effects. GDNF efficiently binds to the GPI-linked GDNF family receptor α1 (GFRα1).^{1, 2} It has been shown that the GDNF/GFRα1 complex can activate not only the canonical GDNF receptor Ret, a receptor tyrosine kinase which signals through the sarcoma protein (Src)/rat sarcoma (Ras)/mitogen-activated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt, NF-κB (nuclear factor ''kappa-light-chain-enhancer'' of activated B cells), JNK (c-Jun N-terminal kinases) and PLCγ (phospholipase γ) pathway, but also with other signaling inducing receptors.^{1, 2, 4, 5, 13} So far, at least four alternative GDNF receptors have been described which are all expressed in midbrain dopaminergic neurons, NCAM,^{14, 15} the integrins αV and βI,^{14, 16} syndecan 3¹⁷ and N-cadherin.¹⁸ Interestingly, Ret is not essential during pre- and postnatal development of the mouse dopaminergic system,^{19, 20, 21, 22, 23} but specifically required for the maintenance of SNpc dopaminergic neurons and their striatal innervation in aged mice.^{23, 24, 25} In contrast, GDNF seems most likely under physiological conditions to be dispensable during development and maintenance of midbrain dopaminergic neurons in mice, although conflicting results exist.^{26, 27, 28} Thus, Ret might be activated by a GDNF-independent mechanism to stimulate SNpc dopaminergic neuron survival. In addition, the in vivo function of the alternative GDNF receptors in the dopaminergic system under physiological and pathophysiological conditions, like PD, and their dependence on GDNF has not yet been addressed in detail. This raised the important question which GDNF receptor might be required to mediate GDNF''s reported neuroprotective and regenerative effect in the dopaminergic system in PD animal models and potentially in PD patients.^{5, 29}Previously, we showed in dopaminergic neuron-specific Ret knockout mice that Ret receptor loss does not result in a higher vulnerability of midbrain dopaminergic neurons against MPTP but to less resprouting of left over dopaminergic neuron axons in the striatum after MPTP intoxication.³⁰ In adult mice endogenous GDNF levels are rather low.^{26, 31} Therefore, we could not rule out in that study the possibility, that higher levels of GDNF—as also used in the clinical GDNF trials in PD patients—might have neuroprotective and regenerating effects even in the absence of the Ret receptor. Here we addressed now this question by viral overexpression of GDNF in MPTP-treated mice lacking expression of Ret again specifically in dopaminergic neurons.^{23, 30} We found that in the absence of Ret in dopaminergic neurons even a substantial overexpression of GDNF in the striatum does not have a neuroprotective and regenerative effect. Thus, despite the expression of alternative GDNF receptors on midbrain dopaminergic neurons, the presence of the canonical GDNF receptor Ret seems to be mandatory for mediating GDNF''s beneficial survival and axonal resprouting effect in these neurons.     

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore.Mitochondrial permeabilization during apoptosis is regulated by the Bcl-2 family of proteins.^{1, 2, 3} Although the Bcl-2 homology 3 (BH3)-only members such as Bid and Bim trigger apoptosis by binding to other family members, the prosurvival members block apoptosis by sequestering their pro-apoptotic relatives. Two remaining members, Bak and Bax, form the apoptotic pore within the mitochondrial outer membrane (MOM).Bak and Bax are globular proteins comprising nine α-helices.^{4, 5} They are activated by BH3-only proteins binding to the α2–α5 surface groove,^{6, 7, 8, 9, 10, 11, 12} or for Bax, to the α1/α6 ‘rear pocket''.¹³ Binding triggers dissociation of the latch domain (α6–α8) from the core domain (α2–α5), together with exposure of N-terminal epitopes and the BH3 domain.^{6, 7, 14, 15, 16} The exposed BH3 domain then binds to the hydrophobic groove in another Bak or Bax molecule to generate symmetric homodimers.^{6, 7, 14, 17, 18} In addition to dimerizing, parts of activated Bak and Bax associate with the lipid bilayer.¹⁹ In Bax, the α5 and α6 helices may insert into the MOM,²⁰ although recent studies indicate that they lie in-plane on the membrane surface, with the hydrophobic α5 sandwiched between the membrane and a BH3:groove dimer interface.^{7, 21, 22, 23} The dimers can be linked via cysteine residues placed in α6,^{18, 24, 25} and more recently via cysteine residues in either α3 or α5,^{6, 21} allowing detection of the higher-order oligomers associated with pore formation.^{26, 27} However, whether these interactions are required for high-order oligomers and pore formation remains unclear.Like most Bcl-2 members, Bak and Bax are targeted to the MOM via a hydrophobic C-terminal region. The C terminus targets Bak to the MOM in healthy cells,²⁸ whereas the Bax C terminus is either exposed²⁹ or sequestered within the hydrophobic groove until apoptotic signals trigger Bax translocation.^{5, 30, 31} The hydrophobic stretch is important, as substituting polar or charged residues decreased targeting of Bak and Bax.^{10, 32} Mitochondrial targeting is also controlled by basic residues at the far C termini,^{32, 33, 34} and by interaction with VDAC2^{35, 36} via the Bak and Bax C termini.^{37, 38} Retrotranslocation of Bak and Bax was also altered by swapping the C termini.³⁹The membrane topology of the Bak and Bax C termini before and after apoptosis has not been examined directly, due in part to difficulty in reconstituting oligomers of full-length Bak in artificial membranes. Nor is it known whether the C termini contribute to pore formation by promoting oligomerization or disturbing the membrane. To address these questions synthetic peptides based on the Bak and Bax C termini have been studied in model membranes. The peptides adopt a predominantly α-helical secondary structure,^{40, 41, 42, 43} with orientation affected by lipid composition.^{42, 44, 45} The peptides could also permeabilize lipid vesicles,^{41, 43, 46, 47} suggesting that the C termini in full-length Bak and Bax may contribute to pore formation.Here we examined the membrane topology of the C termini within full-length Bak and Bax in the MOM, both before and after apoptotic pore formation. After pore formation the α9 helices of Bak (and of Bax) became juxtaposed but did not line the surface of a pore. The α9:α9 interaction occurred after Bak activation and conformation change, but was promoted by formation of BH3:groove dimers. Combining linkage at more than one interface indicated that the Bak α9:α9 interface can link BH3:groove dimers to high-order oligomers, and moreover, that the α6–α9 region is flexible in oligomerized Bak.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

Metabolic Syndrome and Coronary Artery Disease in Ossabaw Compared with Yucatan Swine

Metabolic syndrome (MetS), a compilation of associated risk factors, increases the risk of type 2 diabetes and coronary artery disease (CAD, atherosclerosis), which can progress to the point of artery occlusion. Stents are the primary interventional treatment for occlusive CAD, and patients with MetS and hyperinsulinemia have increased restenosis. Because of its thrifty genotype, the Ossabaw pig is a model of MetS. We tested the hypothesis that, when fed high-fat diet, Ossabaw swine develop more features of MetS, greater native CAD, and greater stent-induced CAD than do Yucatan swine. Animals of each breed were divided randomly into 2 groups and fed 2 different calorie-matched diets for 40 wk: control diet (C) and high-fat, high-cholesterol atherogenic diet (H). A bare metal stent was placed in the circumflex artery, and pigs were allowed to recover for 3 wk. Characteristics of MetS, macrovascular and microvascular CAD, in-stent stenosis, and Ca²⁺ signaling in coronary smooth muscle cells were evaluated. MetS characteristics including, obesity, glucose intolerance, hyperinsulinemia, and elevated arterial pressure were elevated in Ossabaw swine compared to Yucatan swine. Ossabaw swine with MetS had more extensive and diffuse native CAD and in-stent stenosis and impaired coronary blood flow regulation compared with Yucatan. In-stent atherosclerotic lesions in Ossabaw coronary arteries were less fibrous and more cellular. Coronary smooth muscle cells from Ossabaw had impaired Ca²⁺ efflux and intracellular sequestration versus cells from Yucatan swine. Therefore, Ossabaw swine are a superior model of MetS, subsequent CAD, and cellular Ca²⁺ signaling defects, whereas Yucatan swine are leaner and relatively resistant to MetS and CAD.Abbreviations: CAD, coronary artery disease; CSM, coronary smooth muscle; IVGTT, intravenous glucose tolerance test; MetS, metabolic syndrome; SERCA, sarco–endoplasmic reticulum Ca²⁺ ATPase; ET1, endothelin 1; SOCE, store-operated Ca²⁺ entryAtherosclerotic coronary artery disease (CAD) is increased at least 2-fold in patients with metabolic syndrome (MetS)²⁷ and is accompanied by marked microvascular dysfunction that further impairs coronary blood flow.¹⁰ MetS generally is diagnosed by the presence of 3 or more of the following conditions: obesity, insulin resistance, glucose intolerance, dyslipidemia, and hypertension.^17,28 There is strong support for the role of the hyperinsulinemia component of MetS in increased restenosis after percutaneous coronary interventions.^74,75,84,85 Further, our group has shown that severe coronary microvascular dysfunction occurs in MetS.⁵ Because MetS (so-called ‘prediabetes’) affects as much as 27% of the United States population, is increasing dramatically in prevalence,⁹⁴ and can progress to type 2 diabetes, there is great need for basic research using animal models that accurately mimic MetS and the accompanying CAD. Clearly, there is need for study of MetS-induced CAD and in-stent stenosis and the underlying cellular and molecular mechanisms.Mice, rats, and swine are known to recapitulate MetS;^{3,12,36,60,71,72} however, none of these models fully reproduce the combined symptoms of MetS and CAD. Further, transgenic mouse models are simply not adequate for coronary vascular interventions using stents identical to those used in humans,^{18,23,38,55,57,79,83,86} a step that is essential for translation to the clinic. Yucatan and domestic swine are commonly used large animal models for study of cardiovascular disease due to their ability to mimic the neointimal formation and thrombosis observed in humans.⁸⁶ For example, several laboratories have produced severe CAD in swine,^{8,24,51,61,62,68,91} but through toxin-induced pancreatic β-cell ablation and feeding of an atherogenic diet, rather than as a natural development subsequent to MetS or diabetes. Currently, there is a paucity of large animal models that reproduce MetS and CAD.³Research on the obesity-prone Ossabaw miniature swine⁵⁹ clearly indicates that these animals develop MetS and cardiovascular disease when fed a high-calorie atherogenic diet,^{4,5,9,16,19,42,50,52,83,92} Female Ossabaw swine on this type of diet nearly doubled their percentage body fat in only 9 wk, showed insulin resistance, impaired glucose tolerance, dyslipidemia (profound increase in the ratio of low-density to high-density lipoprotein cholesterol, hypertriglyceridemia), hypertension, and early coronary atherosclerosis.¹⁶ These data contrast with those from male Yucatan miniature pigs, which did not develop MetS even after 20 wk on a comparable excess calorie atherogenic diet.^8,68,95 Yucatan swine do not develop MetS through diet manipulation, unlike Ossabaw swine, which consistently recapitulate all MetS characteristics. However, important differences in study design have not allowed direct comparison between Yucatan and Ossabaw swine.Cytosolic Ca²⁺ signaling is involved in ‘phenotypic modulation’ of coronary smooth muscle (CSM), as characterized by proliferation and migration in several in vitro cell culture models^33,35,89,90 and in vivo rodent models of the peripheral circulation (for example, reference 51). The Yucatan swine model of diabetic dyslipidemia shows altered Ca²⁺ extrusion,⁹⁶ Ca²⁺ sequestration by the sarcoplasmic reticulum,^32,34,98 and Ca²⁺ influx through voltage-gated Ca²⁺ channels.⁹⁸ Currently, Ca²⁺ signaling has not been compared directly between MetS Ossabaw and Yucatan swine CSM. Therefore, the purpose of the present study was to test the hypothesis that compared with Yucatan swine on calorie-matched standard chow (for example, Yucatan maintenance diet^8,95) and atherogenic diets, Ossabaw swine have a greater propensity to MetS and CAD with impaired coronary microvascular dysfunction and Ca²⁺ handling in CSM.     

Depletion of white adipocyte progenitors induces beige adipocyte differentiation and suppresses obesity development

Overgrowth of white adipose tissue (WAT) in obesity occurs as a result of adipocyte hypertrophy and hyperplasia. Expansion and renewal of adipocytes relies on proliferation and differentiation of white adipocyte progenitors (WAP); however, the requirement of WAP for obesity development has not been proven. Here, we investigate whether depletion of WAP can be used to prevent WAT expansion. We test this approach by using a hunter-killer peptide designed to induce apoptosis selectively in WAP. We show that targeted WAP cytoablation results in a long-term WAT growth suppression despite increased caloric intake in a mouse diet-induced obesity model. Our data indicate that WAP depletion results in a compensatory population of adipose tissue with beige adipocytes. Consistent with reported thermogenic capacity of beige adipose tissue, WAP-depleted mice display increased energy expenditure. We conclude that targeting of white adipocyte progenitors could be developed as a strategy to sustained modulation of WAT metabolic activity.Obesity, a medical condition predisposing to diabetes, cardiovascular diseases, cancer, and complicating other life-threatening diseases, is becoming an increasingly important social problem.^{1, 2, 3} Development of pharmacological approaches to reduction of body fat has remained a daunting task.⁴ Approved obesity treatments typically produce only moderate and temporary effects.^2,5 White adipocytes are the differentiated cells of white adipose tissue (WAT) that store triglycerides in lipid droplets.^6,7 In contrast, adipocytes of brown adipose tissue (BAT) dissipate excess energy through adaptive thermogenesis. Under certain conditions, white adipocytes can become partially replaced with brown-like ‘beige'' (‘brite'') adipocytes that simulate the thermogenic function of BAT adipocytes.^7,8 Obesity develops in the context of positive energy balance as a result of hypertrophy and hyperplasia of white adipocytes.⁹Expansion and renewal of the white adipocyte pool in WAT continues in adulthood.^10,11 This process is believed to rely on proliferation and self-renewal of mesenchymal precursor cells¹² that we term white adipocyte progenitors (WAPs). WAPs reside within the population of adipose stromal cells (ASCs)¹³ and are functionally similar to bone marrow mesenchymal stem cells (MSCs).^{14, 15, 16} ASCs can be isolated from the stromal/vascular fraction (SVF) of WAT based on negativity for hematopoietic (CD45) and endothelial (CD31) markers.^17,18 ASCs support vascularization as mural/adventitial cells secreting angiogenic factors^5,19 and, unlike bone marrow MSCs, express CD34.^19,20 WAPs have been identified within the ASC population based on expression of mesenchymal markers, such as platelet-derived growth factor receptor-β (PDGFRβ, aka CD140b) and pericyte markers.^17,18 Recently, a distinct ASC progenitor population capable of differentiating into both white and brown adipocytes has been identified in WAT based on PDGFRα (CD140a) expression and lack of PDGFRβ expression.^21,22 The physiological relevance of the two precursor populations residing in WAT has not been explored.We have previously established an approach to isolate peptide ligands binding to receptors selectively expressed on the surface of cell populations of interest.^{23, 24, 25, 26, 27} Such cell-targeted peptides can be used for targeted delivery of experimental therapeutic agents in vivo. A number of ‘hunter-killer'' peptides²⁸ composed of a cell-homing domain binding to a surface marker and of KLAKLAK₂ (sequence KLAKLAKKLAKLAK), a moiety inducing apoptosis upon receptor-mediated internalization, has been described by our group.^26,29 Such bimodal peptides have been used for depletion of malignant cells and organ-specific endothelial cells in preclinical animal models.^26,30,31 Recently, we isolated a cyclic peptide WAT7 (amino acid sequence CSWKYWFGEC) based on its specific binding to ASCs.²⁰ We identified Δ-decorin (ΔDCN), a proteolytic cleavage fragment of decorin, as the WAT7 receptor specifically expressed on the surface of CD34+PDGFRβ+CD31-CD45- WAPs and absent on MSCs in other organs.²⁰Here, we investigated whether WAPs are required for obesity development in adulthood. By designing a new hunter-killer peptide that directs KLAKLAK₂ to WAPs through WAT7/ΔDCN interaction, we depleted WAP in the mouse diet-induced obesity model. We demonstrate that WAP depletion suppresses WAT growth. We show that, in response to WAP deficiency, WAT becomes populated with beige adipocytes. Consistent with the reported thermogenic function of beige adipocytes,^32,33 the observed WAT remodeling is associated with increased energy expenditure. We identify a population of PDGFRα-positive, PDGFRβ-negative ASCs reported recently²² as a population surviving WAP depletion and responsible for WAT browning.     

Use of Fat-Fed Rats to Study the Metabolic and Vascular Sequelae of Obesity and ��-Adrenergic Antagonism

Obesity-associated cardiovascular disease exerts profound human and monetary costs, creating a mounting need for cost-effective and relevant in vivo models of the complex metabolic and vascular interrelationships of obesity. Obesity is associated with endothelial dysfunction and inflammation. Free fatty acids (FFA), generated partly through β-adrenergic receptor-mediated lipolysis, may impair endothelium-dependent vasodilation (EDV) by proinflammatory mechanisms. β-Adrenergic antagonists protect against cardiovascular events by mechanisms not fully defined. We hypothesized that β antagonists may exert beneficial effects, in part, by inhibiting lipolysis and reducing FFA. Further, we sought to evaluate the fat-fed rat as an in vivo model of obesity-induced inflammation and EDV. Control and fat-fed rats were given vehicle or β antagonist for 28 d. Serum FFA were measured to determine the association to serum IL6, TNFα, and C-reactive protein and to femoral artery EDV. Compared with controls, fat-fed rats weighed more and had higher FFA, triglyceride, leptin, and insulin levels. Unexpectedly, in control and fat-fed rats, β antagonism increased FFA, yet inflammatory cytokines were reduced and EDV was preserved. Therefore, reduction of FFA is unlikely to be the mechanism by which β antagonists protect the endothelium. These results reflect the need for validation of ex vivo models of obesity-induced inflammation and endothelial dysfunction, concurrent with careful control of dietary fat composition and treatment duration.Abbreviations: CRP, C-reactive protein; EDV, endothelium-dependent vasodilation; FFA, free fatty acids; FTI, flow–time integral; L-NAME, N_ω-nitro-L-arginine methyl ester; MAP, mean arterial pressure; PKA, protein kinase AThe prevalence of overweight and obese adults in the United States has increased by almost 20% over the last 3 decades.³⁶ Similar upward trends have been observed in persons between 6 and 19 y of age.³⁷ The obesity epidemic extracts a monetary cost of more than $92 billion on medical care alone⁵⁶ and a profound human price in the form of increased disease³⁵ and higher death rates.⁵⁰Obese adults have a higher risk of morbidity and mortality due to cardiovascular disease.⁵⁷ In health, endothelial cells that line the luminal surface of blood vessels release mediators that facilitate the appropriate regulation of multiple processes, including vascular permeability, inflammation and cell adhesion, coagulation, maintenance of intercellular matrix, lipid metabolism, and vascular reactivity.^25,42 Dysregulation of these processes favors inflammation, coagulation, and vasoconstriction. Not surprisingly, endothelial dysfunction as measured by impairment of endothelium-dependent vasodilation (EDV) is an early and reliable predictor of cardiovascular events in humans.^43,47Obese persons have increased serum free fatty acids (FFA).¹ Obesity^14,53 and FFA¹³ are associated with increased circulating inflammatory markers, specifically IL6, TNFα, and C-reactive protein (CRP). In addition, both obesity³² and FFA^8,46 are associated with impaired EDV, and FFA exert direct adverse inflammatory effects on the endothelium.¹⁸Partly in response to stimulation of β-adrenergic receptors, FFA are the principle moiety secreted from adipocytes. β-Adrenergic antagonist drugs reduce morbidity and mortality in patients with coronary artery disease^9,26 and affect both the myocardium^2,38 and vasculature.^5,51,55 The mechanisms by which β-adrenergic antagonists exert protection remain unclear, but reduced generation of FFA might play a role.The first aim of the present study was to determine whether β antagonism lowers serum FFA in fat-fed rats and whether the magnitude and direction of change in FFA is correlated with circulating inflammatory markers and EDV. The β₁- and β₃-receptor subtypes predominantly mediate lipolysis in rodent adipose;^12,30 however, to minimize the potential for compensatory upregulation of unopposed receptors in this study, the β₁β₂ antagonist propranolol was combined with the β₃ antagonist SR59230A (Sigma-Aldrich, St Louis, MO) to exert antagonism at all 3 receptor subtypes. To test whether the high-fat dietary treatment was associated with a metabolic milieu consistent with obesity, serum triglycerides, leptin, glucose, and insulin concentrations were measured.An inexpensive, valid, and physiologically relevant in vivo system would be valuable for studying the pandemic of obesity³¹ and related endothelial dysfunction.¹⁷ We are unaware of studies of whether long-term high-fat feeding affects in vivo EDV in the rat, although a study validating the use of high-resolution ultrasonography to measure in vivo flow-mediated vasodilation in normal rats was published recently.¹⁷ The second aim of our study was to investigate the fat-fed rat as a model of human diet-induced endothelial dysfunction. To retain the complex metabolic-vascular interplay that occurs in the intact organism, we used in vivo measures of EDV to assess the integrated physiologic response. In humans, the dilator response of peripheral vessels is associated with coronary EDV response.⁴⁸ We studied the rat femoral artery, with the aim of demonstrating changes in vasodilator responses in this easily isolated peripheral vascular bed.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.     

Naturally Occurring Disseminated Group B Streptococcus Infections in Postnatal Rats

Group B Streptococcus (Streptococcus agalactiae, GBS) is a gram-positive commensal and occasional opportunistic pathogen of the human vaginal, respiratory, and intestinal tracts that can cause sepsis, pneumonia, or meningitis in human neonates, infants, and immunosuppressed persons. We report here on a spontaneous outbreak of postnatal GBS-associated disease in rats. Ten of 26 (38.5%) 21- to 24-d-old rat pups died or were euthanized due to a moribund state in a colony of rats transgenic for the human diphtheria toxin receptor on a Munich–Wistar–Frömter genetic background. Four pups had intralesional coccoid bacteria in various organs without accompanying inflammation. GBS was isolated from the liver of 2 of these pups and from skin abscesses in 3 littermates. A connection with the transgene could not be established. A treatment protocol was evaluated in the remaining breeding female rats. GBS is a potentially clinically significant spontaneous infection in various populations of research rats, with some features that resemble late-onset postnatal GBS infection in human infants.Abbreviations: GBS, Group B Streptococcus; MWF, Munich Wistar Frömter; hDTR, human diphtheria toxin receptorStreptococci are gram-positive, coccoid bacteria that typically are classified according to their hemolytic capacity. α-hemolytic streptococci produce a zone of partial hemolysis that appears greenish on blood agar, whereas β-hemolytic streptococci produce a zone of complete hemolysis, and γ-hemolytic organisms produce no hemolysis on blood agar.²⁴ The β-hemolytic streptococci are further subdivided into Lancefield groups (A through G), according to cell-wall carbohydrate antigens.^24,29,39 The group B β-hemolytic Streptococcus (GBS) have been speciated as Streptococcus agalactiae.^28,39 It was first isolated as a causative agent of mastitis in cattle.²⁹ This organism has since been recognized as a cause of severe infection in human neonates.^28,39 In humans, GBS is harbored asymptomatically in the maternal genitourinary tract.^24,28 Infants can be infected and present with serious systemic disease in the first week of life (early-onset GBS) or from 1 wk to 3 mo of age (late-onset GBS).³⁹ In laboratory animals, rats have been used experimentally as models for neonatal^{1,6,7,20,37,38,43,44,47,50,51} or adult⁴⁵ GBS infection, but to our knowledge, GBS has not been associated with spontaneous disease in rats.