Mary Susan Burnett

Neuropeptide Y acts directly in the periphery on fat tissue and mediates stress-induced obesity and metabolic syndrome.

The relationship between stress and obesity remains elusive. In response to stress, some people lose weight, whereas others gain. Here we report that stress exaggerates diet-induced obesity through a peripheral mechanism in the abdominal white adipose tissue that is mediated by neuropeptide Y (NPY). Stressors such as exposure to cold or aggression lead to the release of NPY from sympathetic nerves, which in turn upregulates NPY and its Y2 receptors (NPY2R) in a glucocorticoid-dependent manner in the abdominal fat. This positive feedback response by NPY leads to the growth of abdominal fat. Release of NPY and activation of NPY2R stimulates fat angiogenesis, macrophage infiltration, and the proliferation and differentiation of new adipocytes, resulting in abdominal obesity and a metabolic syndrome-like condition. NPY, like stress, stimulates mouse and human fat growth, whereas pharmacological inhibition or fat-targeted knockdown of NPY2R is anti-angiogenic and anti-adipogenic, while reducing abdominal obesity and metabolic abnormalities. Thus, manipulations of NPY2R activity within fat tissue offer new ways to remodel fat and treat obesity and metabolic syndrome.

miR‐125a‐5p impairs endothelial cell angiogenesis in aging mice via RTEF‐1 downregulation

Increasing evidence suggests that microRNAs (miRNAs) play important roles in impaired endothelial cell (EC) angiogenesis during aging. However, their exact roles in the aging process remain unclear. We aimed to determine whether miRNAs cause angiogenesis defects in ECs during aging and to uncover the underlying mechanisms. To study the miRNA‐induced changes in ECs during aging, we performed microarray analyses on arterial ECs collected from young and aging mice. Using qRT–PCR, we showed that microRNA‐125a‐5p (mir‐125a‐5p) expression was approximately 2.9 times higher in old endothelial cells (OECs) compared with samples collected from young animals. Western blot assays showed a lower expression level of an mir‐125a‐5p target known as related transcriptional enhancer factor‐1 (RTEF‐1) in OECs compared with its expression levels in young cells. Overexpression of mir‐125a‐5p in young endothelial cells (YECs) using pre‐mir‐125a‐5p caused the downregulation of RTEF‐1, endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) and resulted in impaired angiogenesis, as evidenced by spheroid sprouting and tube formation assays in vitro. Conversely, repression of mir‐125a‐5p in OECs using anti‐mir‐125a‐5p increased RTEF‐1, eNOS and VEGF expression and improved EC angiogenesis. Importantly, impaired angiogenesis caused by knock‐down of RTEF‐1 was not efficiently rescued by anti‐mir‐125a‐5p. Dual‐luciferase reporter gene analysis showed that RTEF‐1 is a direct target of mir‐125a‐5p, which regulates angiogenesis by repressing RTEF‐1 expression and modulating eNOS and VEGF expression. These findings indicate that mir‐125a‐5p and RTEF‐1 are potential therapeutic targets for improving EC‐mediated angiogenesis in elderly individuals.

Of Mice and Men: Neuropeptide Y and Its Receptors Are Associated with Atherosclerotic Lesion Burden and Vulnerability

Neuropeptide Y (NPY), a sympathetic and platelet-derived vascular mitogen and angiogenic factor, has been implicated in atherosclerosis in animal and human genetic studies. Here we evaluate its association with human and murine atherosclerosis, and assess the role of platelet-derived NPY in lesion vulnerability. NPY immunoreactivity (NPY-ir) was measured in the platelet-poor and platelet-rich (PRP) plasmas, and NPY receptors (mitogenic Y1R and angiogenic Y2 and Y5Rs), CD26/DPPIV (a protease forming Y2/Y5-selective agonist), CD31-positive vascularity, and lesion morphology assessed by histo- and immunocyto-chemistry—in patients with peripheral artery disease (PAD) and healthy volunteers, and in lard-fed ApoE−/− mice. NPY and NPY-R immunostaining was greater in lesions from PAD patients compared to normal vessels of healthy volunteers (pu2009<u20090.001), and localized to smooth muscle cells, macrophages, and adventitial/neovascular endothelial cells. CD26/DPPIV staining co-localized with CD31-positive endothelial cells only in atherosclerotic lesions. NPY-ir in PRP (but not plasma) and vascular immunostaining was higher (pu2009<u20090.05 and 0.001, respectively) in men (not women) with PAD compared to healthy subjects. A similar gender specificity was observed in mice. PRP NPY-ir levels correlated with lesion area (pu2009=u20090.03), necrotic core area, and the necrotic core-to-lesion area ratio (pu2009<u20090.01) in male, but not female, mice. Also males with neovascularized lesions had higher PRP NPY-ir levels than those lacking lesion microvessels (pu2009<u20090.05). NPY and its Rs are up-regulated in human and murine atherosclerotic lesions suggesting pathogenic role. DPPIV expression by microvascular endothelium in atherosclerotic tissue may shift NPY’s affinity toward angiogenic Y2/Y5Rs, and thus enhance angiogenesis and lesion vulnerability. Remarkably, plaque neovascularization was associated with increased NPY-ir in PRP in males but not females, suggesting that platelet NPY may be a novel mediator/marker of lesion vulnerability particularly in males, for reasons that remain to be determined. Both animal and human data suggest that NPY is an important contributor to, and platelet NPY-ir a marker of, atherosclerotic lesion burden and vulnerability but only in males, perhaps due to androgen-dependent up-regulation of NPY, previously shown in rats.

Aging-Induced Collateral Dysfunction: Impaired Responsiveness of Collaterals and Susceptibility to Apoptosis via Dysfunctional eNOS signaling

Despite positive animal studies, clinical angiogenesis trials have been disappointing, possibly due to risk factors present in humans but usually unexplored in animals. We recently demonstrated aging causes impaired collateral remodeling and collateral dropout; here, we investigate potential mechanisms responsible for these findings. Four-, 10-, and 18-month-C57BL/6J mice were subjected to femoral artery ligation; flow was measured using laser Doppler perfusion imaging. Endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS were measured in calf muscle. Apoptosis was assessed in endothelial (EC) and smooth muscle (SMC) cells isolated from young and old mice. Angiogenesis was measured using a Matrigel plug assay. Lethally irradiated young and old mice received bone marrow cells (BMC) from either young or old donors and were subjected to femoral artery ligation (FAL). BMC mobilization and homing were assessed. Flow recovery was impaired and less eNOS and phosphorylated eNOS was present in older vs. young mice (pu2009<u20090.001 and pu2009=u20090.015, respectively). ECs and SMCs from older mice were more sensitive to an apoptotic stimulus, but were rescued by NO-enhancing drugs. In older mice, angiogenesis (Matrigel plug assay) was impaired, as was mobilization and homing of BM progenitor cells following FAL. Although both mobilization and homing improved when older mice received BMC transplantation from young donors, flow recovery failed to improve. Aging impairs BMC mobilization and homing, collateral responsiveness to angiogenic stimuli, and increases EC and SMC susceptibility to apoptosis via dysfunctional eNOS signaling. The latter could contribute to impaired remodeling and collateral dropout. These finding identify potential obstacles to therapeutic interventions in elderly patients.

A new murine model of stress-induced complex atherosclerotic lesions

SUMMARY The primary purpose of this investigation was to determine whether ApoE−/− mice, when subjected to chronic stress, exhibit lesions characteristic of human vulnerable plaque and, if so, to determine the time course of such changes. We found that the lesions were remarkably similar to human vulnerable plaque, and that the time course of lesion progression raised interesting insights into the process of plaque development. Lard-fed mixed-background ApoE−/− mice exposed to chronic stress develop lesions with large necrotic core, thin fibrous cap and a high degree of inflammation. Neovascularization and intraplaque hemorrhage are observed in over 80% of stressed animals at 20 weeks of age. Previously described models report a prevalence of only 13% for neovascularization observed at a much later time point, between 36 and 60 weeks of age. Thus, our new stress-induced model of advanced atherosclerotic plaque provides an improvement over what is currently available. This model offers a tool to further investigate progression of plaque phenotype to a more vulnerable phenotype in humans. Our findings also suggest a possible use of this stress-induced model to determine whether therapeutic interventions have effects not only on plaque burden, but also, and importantly, on plaque vulnerability.

Platelet neuropeptide Y is critical for ischemic revascularization in mice

We previously reported that the sympathetic neurotransmitter neuropeptide Y (NPY) is potently angiogenic, primarily through its Y2 receptor, and that endogenous NPY is crucial for capillary angiogenesis in rodent hindlimb ischemia. Here we sought to identify the source of NPY responsible for revascularization and its mechanisms of action. At d 3, NPY–/– mice demonstrated delayed recovery of blood flow and limb function, consistent with impaired collateral conductance, while ischemic capillary angiogenesis was reduced (~70%) at d 14. This biphasic temporal response was confirmed by 2 peaks of NPY activation in rats: a transient early increase in neuronally derived plasma NPY and increase in platelet NPY during late‐phase recovery. Compared to NPY‐null platelets, collagen‐activated NPY‐rich platelets were more mitogenic (~2‐fold vs. ~1.6‐fold increase) for human microvascular endothelial cells, and Y2/Y5 receptor antagonists ablated this difference in proliferation. In NPY+/+ mice, ischemic angiogenesis was prevented by platelet depletion and then restored by transfusion of platelets from NPY+/+ mice, but not NPY–/– mice. In thrombocytopenic NPY–/– mice, transfusion of wild‐type platelets fully restored ischemia‐induced angiogenesis. These findings suggest that neuronally derived NPY accelerates the early response to femoral artery ligation by promoting collateral conductance, while platelet‐derived NPY is critical for sustained capillary angiogenesis.—Tilan, J. U., Everhart, L. M., Abe, K., Kuo‐Bonde, L., Chalothorn, D., Kitlinska, J., Burnett, M. S., Epstein, S. E., Faber, J. E., Zukowska, Z. Platelet neuropeptide Y is critical for ischemic revascularization in mice. FASEB J. 27, 2244–2255 (2013). www.fasebj.org

Polymorphisms in dipeptidyl peptidase IV gene are associated with the risk of myocardial infarction in patients with atherosclerosis.

BACKGROUNDnDipeptidyl peptidase IV (DPP-IV) is not only important in pancreatic β-cell regulation but also has proinflammatory actions that can contribute to atherosclerosis progression. Previously, we showed that DPP-IV is co-localized with CD31 (an endothelial cell marker) in the neovessels within the human atherosclerotic plaques. These characteristics of DPP-IV may predispose patients with coronary artery disease (CAD) to plaque rupture and thus to myocardial infarction. The goal of this investigation was to determine whether genetic alterations in DPP-IV predispose to plaque vulnerability and myocardial infarction (MI).nnnMETHODSnBetween Aug 2004, and March 2007, blood samples of patients (age <60) with angiographically documented CAD were collected. Demographic, clinical, risk factor, and angiographic data were recorded. Eight hundred and seventy five patients of European ancestry with angiographic CAD were divided into those with MI (n=421) and those without (n=454). A genome-wide association study was performed using the Affymetrix 6.0 chip to identify loci that predispose to MI. In the current study we only focused on DPP4 gene to assess the association of single nucleotide polymorphisms (SNPs) in the DPP-IV gene and risk of MI in patients with CAD. For genotyped SNPs, association was tested by logistic regression with significance level of 0.05. Plasma DPP-IV level was measured using a commercial ELISA kit.nnnRESULTSnAverage patients age at diagnosis of CAD was 46.8years for MI group and 50.8 in the non MI group. There was no difference in distribution of traditional risk factors between the two groups. We identified one SNP (rs3788979) that was significantly related to angiographic CAD with MI, vs. without MI (OR: 1.36, p=0.03). The association of the identified SNP to MI risk was not attenuated after adjustment for traditional risk factors. The SNP was associated with lower levels of plasma DPP-IV (p=0.005). Moreover, CAD patients with the major alleles (GG) and no MI had highest plasma DPP-IV levels. (481.6, p=0.002).nnnCONCLUSIONSnA polymorphism in the DPP-IV gene in patients with known CAD may increase the risk of MI. This SNP is associated with decreased plasma DPP4 level in patients with MI.

CHRONIC STRESS ACCELERATES AGING INDUCED COLLATERAL DYSFUNCTION IN C57BL6 MOUSE MODEL OF HIND LIMB ISCHEMIA

Aging is a major vascular risk factor. Chronic stress is associated with impaired endothelial function. We hypothesized that chronic stress accelerates aging induced collateral dysfunction in mouse model of hind limb ischemia.nn20 month–old C57Bl6 mice were subjected to chronic stress, either cold