Neuropeptide Y receptor interactions regulate its mitogenic activity.

Neuropeptide Y (NPY) is a multifunctional neurotransmitter acting via G protein-coupled receptors - Y1R, Y2R and Y5R. NPY activities, such as its proliferative effects, are mediated by multiple receptors, which have the ability to dimerize. However, the role of this receptor interplay in NPY functions remains unclear. The goal of the current study was to identify NPY receptor interactions, focusing on the ligand-binding fraction, and determine their impact on the mitogenic activity of the peptide. Y1R, Y2R and Y5R expressed in CHO-K1 cells formed homodimers detectable on the cell surface by cross-linking. Moreover, Y1R and Y5R heterodimerized, while no Y2R/Y5R heterodimers were detected. Nevertheless, Y5R failed to block internalization of its cognate receptor in both Y1R/Y5R and Y2R/Y5R transfectants, indicating Y5R transactivation upon stimulation of the co-expressed receptor. These receptor interactions correlated with an augmented mitogenic response to NPY. In Y1R/Y5R and Y2R/Y5R transfectants, the proliferative response started at picomolar NPY concentrations, while nanomolar concentrations were needed to trigger proliferation in cells transfected with single receptors. Thus, our data identify direct and indirect heterotypic NPY receptor interactions as the mechanism amplifying its activity. Understanding these processes is crucial for the design of treatments targeting the NPY system.

Temporal dissection of tumorigenesis in primary cancers.

Timely intervention for cancer requires knowledge of its earliest genetic aberrations. Sequencing of tumors and their metastases reveals numerous abnormalities occurring late in progression. A means to temporally order aberrations in a single cancer, rather than inferring them from serially acquired samples, would define changes preceding even clinically evident disease. We integrate DNA sequence and copy number information to reconstruct the order of abnormalities as individual tumors evolve for 2 separate cancer types. We detect vast, unreported expansion of simple mutations sharply demarcated by recombinative loss of the second copy of TP53 in cutaneous squamous cell carcinomas (cSCC) and serous ovarian adenocarcinomas, in the former surpassing 50 mutations per megabase. In cSCCs, we also report diverse secondary mutations in known and novel oncogenic pathways, illustrating how such expanded mutagenesis directly promotes malignant progression. These results reframe paradigms in which TP53 mutation is required later, to bypass senescence induced by driver oncogenes.

A genetic risk score combining ten psoriasis risk loci improves disease prediction.

Psoriasis is a chronic, immune-mediated skin disease affecting 2-3% of Caucasians. Recent genetic association studies have identified multiple psoriasis risk loci; however, most of these loci contribute only modestly to disease risk. In this study, we investigated whether a genetic risk score (GRS) combining multiple loci could improve psoriasis prediction. Two approaches were used: a simple risk alleles count (cGRS) and a weighted (wGRS) approach. Ten psoriasis risk SNPs were genotyped in 2815 case-control samples and 858 family samples. We found that the total number of risk alleles in the cases was significantly higher than in controls, mean 13.16 (SD 1.7) versus 12.09 (SD 1.8), p = 4.577×10(-40). The wGRS captured considerably more risk than any SNP considered alone, with a psoriasis OR for high-low wGRS quartiles of 10.55 (95% CI 7.63-14.57), p = 2.010×10(-65). To compare the discriminatory ability of the GRS models, receiver operating characteristic curves were used to calculate the area under the curve (AUC). The AUC for wGRS was significantly greater than for cGRS (72.0% versus 66.5%, p = 2.13×10(-8)). Additionally, the AUC for HLA-C alone (rs10484554) was equivalent to the AUC for all nine other risk loci combined (66.2% versus 63.8%, p = 0.18), highlighting the dominance of HLA-C as a risk locus. Logistic regression revealed that the wGRS was significantly associated with two subphenotypes of psoriasis, age of onset (p = 4.91×10(-6)) and family history (p = 0.020). Using a liability threshold model, we estimated that the 10 risk loci account for only 11.6% of the genetic variance in psoriasis. In summary, we found that a GRS combining 10 psoriasis risk loci captured significantly more risk than any individual SNP and was associated with early onset of disease and a positive family history. Notably, only a small fraction of psoriasis heritability is captured by the common risk variants identified to date.

Green technologies for room temperature nucleic acid storage.

Maintaining the long-term integrity of nucleic acids in the laboratory has traditionally required the use of freezers. However, novel nucleic acid stabilization technologies may allow for the storage of DNA and RNA at room temperature in a cost-effective, environmentally friendly manner. In this study, we evaluated two novel products for room temperature DNA storage: Biomatrica's DNA SampleMatrix technology and GenVault's GenTegra DNA technology. We compared the integrity and quality of DNA stored using these products against DNA stored in a -20 C freezer by performing downstream testing with short range PCR, long range PCR, DNA sequencing, and SNP microarrays. In addition, we tested Biomatrica's RNAstable product for its ability to preserve RNA at room temperature for use in a quantitative reverse transcription PCR assay.

Combining angiogenic gene and stem cell therapies for myocardial infarction.

BACKGROUND: Transplantation of stem cells from various sources into infarcted hearts has the potential to promote myocardial regeneration. However, the regenerative capacity is limited partly as a result of the low survival rate of the transplanted cells in the ischemic myocardium. In the present study, we tested the hypothesis that combining cell and angiogenic gene therapies would provide additive therapeutic effects via co-injection of bone marrow-derived mesenchymal stem cells (MSCs) with an adeno-associated viral vector (AAV), MLCVEGF, which expresses vascular endothelial growth factor (VEGF) in a cardiac-specific and hypoxia-inducible manner. METHODS: MSCs isolated from transgenic mice expressing green fluorescent protein and MLCVEGF packaged in AAV serotype 1 capsid were injected into mouse hearts at the border of ischemic area, immediately after occlusion of the left anterior descending coronary, individually or together. Engrafted cells were detected and quantified by real-time polymerase chain reaction and immunostaining. Angiogenesis and infarct size were analyzed on histological and immunohistochemical stained sections. Cardiac function was analyzed by echocardiography. RESULTS: We found that co-injection of AAV1-MLCVEGF with MSCs reduced cell loss. Although injection of MSCs and AAV1-MLCVEGF individually improved cardiac function and reduced infarct size, co-injection of MSC and AAV1-MLCVEGF resulted in the best improvement in cardiac function as well as the smallest infarct among all groups. Moreover, injection of AAV1-MLCVEGF induced neovasculatures. Nonetheless, injection of MSCs attracted endogenous stem cell homing and increased scar thickness. CONCLUSIONS: Co-injection of MLCVEGF and MSCs in ischemic hearts can result in better cardiac function and MSC survival, compared to their individual injections, as a result of the additive effects of each therapy.

Additive effect of AAV-mediated angiopoietin-1 and VEGF expression on the therapy of infarcted heart.

Vascular endothelial growth factor (VEGF) is a key angiogenic factor and has been used experimentally for induction of neovasculature in ischemic myocardium. However, blood vessels induced by VEGF are immature. Angiopoietin-1 (ang-1) has the ability to recruit and sustain periendothelial support cells and promote vascular maturation. Thus, co-expression of the two may yield a better result than expression of either one alone. Two adeno-associated viral vectors (AAV), CMVVEGF and CMVang-1 with the CMV promoter driving VEGF or ang-1 gene expression, respectively, were injected into ischemic mouse hearts individually or together in different ratios. The results show that co-injected groups had more capillaries than the CMVang-1 group and similar densities of capillaries and alpha-actin positive vessels as the CMVVEGF group. Neovasculature induced by CMVVEGF was leaky. In contrast, neovasculature in CMVang-1-injected or CMVVEGF and CMVang-1 co-injected hearts was less leaky than that in CMVVEGF-injected hearts. The group that received CMVang-1 and CMVVEGF in a 1:1 ratio had the smallest infarct size and best cardiac function and regional wall movement among all the groups. We conclude that ang-1 and VEGF can compensate for each others' shortcomings and yield a better therapeutic effect by acting together.

VEGF improves survival of mesenchymal stem cells in infarcted hearts.

Bone marrow-derived mesenchymal stem cells (MSC) are a promising source for cell-based treatment of myocardial infarction (MI), but existing strategies are restricted by low cell survival and engraftment. We examined whether vascular endothelial growth factor (VEGF) improve MSC viability in infarcted hearts. We found long-term culture increased MSC-cellular stress: expressing more cell cycle inhibitors, p16(INK), p21 and p19(ARF). VEGF treatment reduced cellular stress, increased pro-survival factors, phosphorylated-Akt and Bcl-xL expression and cell proliferation. Co-injection of MSCs with VEGF to MI hearts increased cell engraftment and resulted in better improvement of cardiac function than that injected with MSCs or VEGF alone. In conclusion, VEGF protects MSCs from culture-induce cellular stress and improves their viability in ischemic myocardium, which results in improvements of their therapeutic effect for the treatment of MI.

Interactions of multiple signaling pathways in neuropeptide Y-mediated bimodal vascular smooth muscle cell growth.

Neuropeptide Y (NPY), a sympathetic cotransmitter, acts via G protein-coupled receptors to stimulate constriction and vascular smooth muscle cell (VSMC) proliferation through interactions with its Y1 receptors. However, VSMC proliferation appears bimodal, with high- and low-affinity peaks differentially blocked by antagonists of both Y1 and Y5 receptors. Here, we sought to determine the signaling mechanisms of NPY-mediated bimodal mitogenesis. In rat aortic VSMCs, NPY's mitogenic effect at all concentrations was blocked by pertussis toxin and was associated with decreased forskolin-stimulated cAMP levels. NPY also increased intracellular calcium levels; in contrast to mitogenesis, this effect was dose dependent. The rise in intracellular Ca2+ depended on extracellular Ca2+ and was mediated via activation of Y1 receptors, but not Y5 receptors. Despite differences in calcium, the signaling pathways activated at low and high NPY concentrations were similar. The mitogenic effect of the peptide at all doses was completely blocked by inhibitors of calcium/calmodulin-dependent kinase II (CaMKII), protein kinase C (PKC), and mitogen-activated protein kinase kinase, MEK1/2. Thus, in VSMCs, NPY-mediated mitogenesis signals primarily via Y1 receptors activating 2 Ca2+-dependent, growth-promoting pathways -- PKC and CaMKII. At the high-affinity peak, these 2 pathways are amplified by Y5 receptor-mediated, calcium-independent inhibition of the adenylyl cyclase - protein kinase A (PKA) pathway. All 3 mechanisms converge to the extracellular signal-regulated kinases (ERK1/2) signaling cascade and lead to VSMC proliferation.

Differential effects of neuropeptide Y on the growth and vascularization of neural crest-derived tumors.

Neuropeptide Y (NPY) is a sympathetic neurotransmitter recently found to be potently angiogenic and growth promoting for endothelial, vascular smooth muscle and neuronal cells. NPY and its cognate receptors, Y1, Y2 and Y5, are expressed in neural crest-derived tumors; however, their role in regulation of growth is unknown. The effect of NPY on the growth and vascularization of neuroendocrine tumors was tested using three types of cells: neuroblastoma, pheochromocytoma, and Ewing's sarcoma family of tumors (ESFT). The tumors varied in expression of NPY receptors, which was linked to differential functions of the peptide. NPY stimulated proliferation of neuroblastoma cells via Y2/Y5Rs and inhibited ESFT cell growth by Y1/Y5-mediated apoptosis. In both tumor types, NPY receptor antagonists altered basal growth levels, indicating a regulatory role of autocrine NPY. In addition, the peptide released from the tumor cells stimulated endothelial cell proliferation, which suggests its paracrine angiogenic effects. In nude mice xenografts, exogenous NPY stimulated growth of neuroblastoma tumors, whereas it increased apoptosis and reduced growth of ESFT. However, in both tumors, NPY treatment led to an increase in tumor vascularization. Taken together, this is the first report of NPY being a growth-regulatory factor for neuroendocrine tumors, acting both by autocrine activation of tumor cell proliferation or apoptosis and by angiogenesis. NPY and its receptors may become targets for novel approaches in the treatment of these diseases, directed against both tumor cell proliferation and angiogenesis.

Neuropeptide Y: multiple receptors and multiple roles in cardiovascular diseases.

Neuropeptide Y (NPY), a sympathetic co-transmitter, acts through multiple G protein-coupled receptors (Y1 to y6) to elicit its vast range of effects in the cardiovascular, immune, and central and peripheral nervous systems. Initially, the focus of the function of NPY in the cardiovascular system involved its acute actions, such as vasoconstriction via the Y1 receptor. However, recent studies have shown that NPY is a potent growth and angiogenic factor, which acts on multiple receptor subtypes. To be more specific, NPY-mediated vascular smooth muscle cell growth, leading to neointima formation, involves Y1 and Y1 receptors, while the angiogenic effects of NPY include Y2 and Y5 receptor activation. The presence of dipeptidyl peptidase IV also influences the cardiovascular responses of NPY by acting as a converting enzyme, shifting NPY activities away from Y1. Thus, agonists and antagonists aimed at the NPY system represent a new avenue for drug treatment, which may help alleviate several cardiovascular disorders in which vascular remodeling plays a major role, such as atherosclerosis, restenosis following balloon angioplasty, hypertension and peripheral vascular disease.

Neuropeptide Y: a new mediator linking sympathetic nerves, blood vessels and immune system?

Neuropeptide Y (NPY(1-36)), a sympathetic cotransmitter and neurohormone, has pleiotropic activities ranging from the control of obesity to anxiolysis and cardiovascular function. Its actions are mediated by multiple Gi/o-coupled receptors (Y1-Y5) and modulated by dipeptidyl peptidase IV (DPPIV/cd26), which inactivates NPY's Y1-agonistic activity but generates the Y2 and Y5-agonist, NPY(3-36). Released by sympathetic activity, NPY is a major mediator of stress, responsible for prolonged vasoconstriction via Y1 receptors. Y1 receptors also mediate NPY's potent vascular growth-promoting activity leading in vivo in rodents to neointima formation. This and the association of a polymorphism of the NPY signal peptide with increased lipidemia and carotid artery thickening in humans strongly suggest NPY's role in atherosclerosis. NPY and DPPIV/cd26 are also coexpressed in the endothelium, where the peptide activates angiogenesis. A similar system exists in immune cells, where NPY and DPPIV/cd26 are coactivated and involved in the modulation of cytokine release and immune cell functions. Thus, NPY, both a messenger and a modulator for all three systems, is poised to play an important regulatory role facilitating interactions among sympathetic, vascular and immune systems in diverse pathophysiological conditions such as hypertension, atherosclerosis and stress-related alterations of immunity.

Mitogenic actions of neuropeptide Y in vascular smooth muscle cells: synergetic interactions with the beta-adrenergic system.

Neuropeptide Y (NPY), a sympathetic cotransmitter and vasoconstrictor, also stimulates vascular smooth muscle cell (VSMC) growth, but which of its Y1-Y5 receptors are involved remains unclear. In quiescent rat VSMCs, NPY receptor mRNAs were undetectable (reverse transcription-polymerase chain reaction), but Y1, Y2, and Y5 expression were upregulated or induced following NPY treatment. Concomitantly, NPY increased up to twofold [3H]thymidine incorporation and cell number bimodally, with a high-affinity peak at pM and low affinity peak at nM concentrations. The Y1 or Y5 (not Y2) antagonist alone did not change the high-affinity peak but decreased the low affinity peak by 50% and fully blocked NPY's response when combined. In VSMCs lacking NPY receptors and responsiveness, transient Y1 cDNA transfection restored their mitogenic response (blocked by the Y1 antagonist). In VSMCs with low or no NPY responsiveness, pre-exposure to beta-adrenergic receptor agonist (isoproterenol), forskolin, or dibutyryl cAMP augmented NPY's mitogenic effect, while upregulating Y1, Y2, and Y5 receptor expression (isoproterenol only). Thus, NPY is a potent vascular mitogen acting via Y1 and Y5 receptors. However, since their expression is low in nonproliferating cells, amplification of NPY's mitogenic responses requires upregulation of at least the Y1 receptor by NPY itself or beta-adrenergic, cAMP-dependent activation.

Neuropeptide Y-induced angiogenesis in aging.

Age-related changes in NPY-driven angiogenesis were investigated using Matrigel and aortic sprouting assays in young (2 months.) and aged (18 months.) mice. In both assays, NPY-induced vessel growth decreased significantly with age. In parallel, aged mice showed reduced expression (RT-PCR) of Y2 receptors and the NPY converting enzyme, dipeptidyl peptidase IV (DPPIV), in spleens. Aging of human microvascular endothelial cells in vitro led to a loss of their mitogenic responses to NPY accompanied by a lack of NPY receptor mRNAs. Thus, NPY-dependent angiogenesis is impaired with age, which is associated with a decreased expression of endothelial NPY receptors (Y2) and DPPIV.