Aberrant PGC-1α signaling in a lamb model of persistent pulmonary hypertension of the newborn.

BACKGROUND: Persistent Pulmonary Hypertension of the Newborn (PPHN) is characterized by elevated pulmonary vascular resistance (PVR), resulting in hypoxemia. Impaired angiogenesis contributes to high PVR. Pulmonary artery endothelial cells (PAECs) in PPHN exhibit decreased mitochondrial respiration and angiogenesis. We hypothesize that Peroxisome Proliferator-Activated Receptor Gamma Co-Activator-1α (PGC-1α) downregulation leads to reduced mitochondrial function and angiogenesis in PPHN. METHODS: Studies were performed in PAECs isolated from fetal lambs with PPHN induced by ductus arteriosus constriction, with gestation-matched controls and in normal human umbilical vein endothelial cells (HUVECs). PGC-1α was knocked downed in control lamb PAECs and HUVECs and overexpressed in PPHN PAECs to investigate the effects on mitochondrial function and angiogenesis. RESULTS: PPHN PAECs had decreased PGC-1α expression compared to controls. PGC-1α knockdown in HUVECs led to reduced Nuclear Respiratory Factor-1 (NRF-1), Transcription Factor-A of Mitochondria (TFAM), and mitochondrial electron transport chain (ETC) complexes expression. PGC-1α knockdown in control PAECs led to decreased in vitro capillary tube formation, cell migration, and proliferation. PGC-1α upregulation in PPHN PAECs led to increased ETC complexes expression and improved tube formation, cell migration, and proliferation. CONCLUSION: PGC-1α downregulation contributes to reduced mitochondrial oxidative phosphorylation through control of the ETC complexes, thereby affecting angiogenesis in PPHN. IMPACT: Reveals a novel mechanism for angiogenesis dysfunction in persistent pulmonary hypertension of the newborn (PPHN). Identifies a key mitochondrial transcription factor, Peroxisome Proliferator-Activated Receptor Gamma Co-Activator-1α (PGC-1α), as contributing to the altered adaptation and impaired angiogenesis function that characterizes PPHN through its regulation of mitochondrial function and oxidative phosphorylation. May provide translational significance as this mechanism offers a new therapeutic target in PPHN, and efforts to restore PGC-1α expression may improve postnatal transition in PPHN.

Fetal pulmonary hypertension: dysregulated microRNA-34c-Notch1 axis contributes to impaired angiogenesis in an ovine model.

BACKGROUND: Persistent pulmonary hypertension of the newborn (PPHN) occurs when pulmonary vascular resistance (PVR) fails to decrease at birth. Decreased angiogenesis in the lung contributes to the persistence of high PVR at birth. MicroRNAs (miRNAs) regulate gene expression through transcript binding and degradation. They were implicated in dysregulated angiogenesis in cancer and cardiovascular disease. METHODS: We investigated whether altered miRNA levels contribute to impaired angiogenesis in PPHN. We used a fetal lamb model of PPHN induced by prenatal ductus arteriosus constriction and sham ligation as controls. We performed RNA sequencing of pulmonary artery endothelial cells (PAECs) isolated from control and PPHN lambs. RESULTS: We observed a differentially expressed miRNA profile in PPHN for organ development, cell-cell signaling, and cardiovascular function. MiR-34c was upregulated in PPHN PAECs compared to controls. Exogenous miR34c mimics decreased angiogenesis by control PAEC and anti-miR34c improved angiogenesis of PPHN PAEC in vitro. Notch1, a predicted target for miR-34c by bioinformatics, was decreased in PPHN PAECs, along with Notch1 downstream targets, Hey1 and Hes1. Exogenous miR-34c decreased Notch1 expression in control PAECs and anti-miR-34c restored Notch1 and Hes1 expression in PPHN PAECs. CONCLUSION: We conclude that increased miR-34c in PPHN contributes to impaired angiogenesis by decreasing Notch1 expression in PAECs. IMPACT: Adds a novel mechanism for the regulation of angiogenesis in persistent pulmonary hypertension of the newborn. Identifies non-coding RNAs that are involved in the altered angiogenesis in PPHN and thus the potential for future studies to identify links between known pathways regulating angiogenesis. Provides preliminary data to conduct studies targeting miR34c expression in vivo in animal models of pulmonary hypertension to identify the mechanistic role of miR34c in angiogenesis in the lung vasculature.

Decreased Cyclic Guanosine Monophosphate-Protein Kinase G Signaling Impairs Angiogenesis in a Lamb Model of Persistent Pulmonary Hypertension of the Newborn.

Impaired angiogenesis function in pulmonary artery endothelial cells (PAEC) contributes to persistent pulmonary hypertension of the newborn (PPHN). Decreased nitric oxide (NO) amounts in PPHN lead to impaired mitochondrial biogenesis and angiogenesis in the lung; the mechanisms remain unclear. We hypothesized that decreased cyclic guanosine monophosphate (cGMP)-PKG (protein kinase G) signaling downstream of NO leads to decreased mitochondrial biogenesis and angiogenesis in PPHN. PPHN was induced by ductus arteriosus constriction from 128-136 days' gestation in fetal lambs. Control animals were gestation-matched lambs that did not undergo ductal constriction. PAEC isolated from PPHN lambs were treated with the sGC (soluble guanylate cyclase) activator cinaciguat, the PKG activator 8-bromo-cGMP, or the PDE-V (PDE type V) inhibitor sildenafil. Lysates were immunoblotted for mitochondrial transcription factors and electron transport chain C-I (complex I), C-II, C-III, C-IV, and C-V proteins. The in vitro angiogenesis of PAEC was evaluated by using tube-formation and scratch-recovery assays. cGMP concentrations were measured by using an enzyme immunoassay. Fetal lambs with ductal constriction were given sildenafil or control saline through continuous infusion in utero, and the lung histology, capillary counts, vessel density, and right ventricular pressure were assessed at birth. PPHN PAEC showed decreased mitochondrial transcription factor levels, electron transport chain protein levels, and in vitro tube formation and cell migration; these were restored by cinaciguat, 8-bromo-cGMP, and sildenafil. Cinaciguat and sildenafil increased cGMP concentrations in PPHN PAEC. Radial alveolar and capillary counts and vessel density were lower in PPHN lungs, and the right ventricular pressure and Fulton Index were higher in PPHN lungs; these were improved by in utero sildenafil infusion. cGMP-PKG signaling is a potential therapeutic target to restore decreased mitochondrial biogenesis and angiogenesis in PPHN.

Decreased AMP-activated protein kinase (AMPK) function and protective effect of metformin in neonatal rat pups exposed to hyperoxia lung injury.

We investigated the hypothesis that exposure of lungs at the saccular stage of development to hyperoxia leads to persistent growth arrest and dysfunction of 5'AMP-activated protein kinase (AMPK), a key energy sensor in the cell. We exposed neonatal rat pups from postnatal day 1- day 10 (P1-P10) to ≥90% oxygen or control normoxia. Pups were euthanized at P4 or P10 or recovered in normoxia until euthanasia at P21. Half of the pups in each group received AMPK activator, metformin, or saline intraperitoneally from P1 to P10. Lung histology, morphometric analysis, immunofluorescence, and immunoblots were done for changes in lung structure at P10 and P21 and AMPK function at P4, P10, and P21. Phosphorylation of AMPK (p-AMPK) was decreased in lungs at P10 and P21 in hyperoxia-exposed pups. Metformin increased the levels of p-AMPK and PGC-1α, a downstream AMPK target which regulates mitochondrial biogenesis, at P4, P10, and P21 in hyperoxia pups. Lung ATP levels decreased during hyperoxia and were increased by metformin at P10 and P21. Radial alveolar count and alveolar septal tips were decreased and mean linear intercept increased in hyperoxia-exposed pups at P10 and the changes persisted at P21; these were improved by metformin. Lung capillary number was decreased in hyperoxia-exposed pups at P10 and P21 and was restored by metformin. Hyperoxia leads to impaired AMPK function, energy balance and alveolar simplification. The AMPK activator, metformin improves AMPK function and alveolar and vascular growth in this rat pup model of hyperoxia-induced lung injury.

AMP-Kinase Dysfunction Alters Notch Ligands to Impair Angiogenesis in Neonatal Pulmonary Hypertension.

Decreased angiogenesis contributes to persistent pulmonary hypertension of the newborn (PPHN); mechanisms remain unclear. AMPK (5'AMP activated protein kinase) is a key regulator of cell metabolism. We investigated the hypothesis that a decrease in AMPK function leads to mitochondrial dysfunction and altered balance of notch ligands delta-like 4 (DLL4) and Jagged 1 (Jag1) to impair angiogenesis in PPHN. Studies were done in fetal lambs with PPHN induced by prenatal ductus arteriosus constriction and gestation-matched control lambs. PPHN lambs were treated with saline or AMPK agonist metformin. Angiogenesis was assessed in lungs with micro-computed tomography angiography and histology. AMPK function; expression of mitochondrial electron transport chain (ETC) complex proteins I-V, Dll4, and Jag1; mitochondrial number; and in vitro angiogenesis function were assessed in pulmonary artery endothelial cells (PAEC) from control and PPHN lambs. AMPK function was decreased in PPHN PAEC and lung sections. Expression of mitochondrial transcription factor, PGC-1α, ETC complex proteins I-V, and mitochondrial number were decreased in PPHN. In vitro angiogenesis of PAEC and capillary number and vessel volume fraction in the lung were decreased in PPHN. Expression of DLL4 was increased and Jag1 was decreased in PAEC from PPHN lambs. AMPK agonists A769662 and metformin increased the mitochondrial complex proteins and number, in vitro angiogenesis, and Jag1 levels and decreased DLL4 levels in PPHN PAEC. Infusion of metformin in vivo increased the vessel density in PPHN lungs. Decreased AMPK function contributes to impaired angiogenesis in PPHN by altered balance of notch ligands in PPHN.

Adverse early-life environment impairs postnatal lung development in mice.

BACKGROUND: Fetal growth restriction (FGR) is a major risk factor for bronchopulmonary dysplasia (BPD). Maternal stress and poor diet are linked to FGR. Effect of perinatal stress on lung development remains unknown. OBJECTIVE: Using a murine model of adverse early life environment (AELE), we hypothesized that maternal exposure to perinatal environmental stress and high-fat diet (Western diet) lead to impaired lung development in the offspring. METHODS: Female mice were placed on either control diet or Western diet before conception. Those exposed to Western diet were also exposed to perinatal environmental stress, the combination referred to as AELE. Pups were either euthanized at postnatal day 21 (P21) or weaned to control diet and environment until adulthood (8-14 wk old). Lungs were harvested for histology, gene expression by quantitative RT-PCR, microRNA profiling, and immunoblotting. RESULTS: AELE increased the mean linear intercept and decreased the radial alveolar count and secondary septation in P21 and adult mice. Capillary count was also decreased in P21 and adult mice. AELE lungs had decreased vascular endothelial growth factor A (VEGFA), VEGF receptor 2, endothelial nitric oxide synthase, and hypoxia inducible factor-1α protein levels and increased expression of genes that regulate DNA methylation and upregulation of microRNAs that target genes involved in lung development at P21. CONCLUSION: AELE leads to impaired lung alveolar and vascular growth, which persists into adult age despite normalizing the diet and environment at P21. AELE also alters the expression of genes involved in lung remodeling.

Dynamic Phosphorylation of the C Terminus of Hsp70 Regulates the Mitochondrial Import of SOD2 and Redox Balance.

The import of superoxide dismutase-2 (SOD2) into mitochondria is vital for the survival of eukaryotic cells. SOD2 is encoded within the nuclear genome and translocated into mitochondria for activation after translation in the cytosol. The molecular chaperone Hsp70 modulates SOD2 activity by promoting import of SOD2 into mitochondria. In turn, the activity of Hsp70 is controlled by co-chaperones, particularly CHIP, which directs Hsp70-bound proteins for degradation in the proteasomes. We investigated the mechanisms controlling the activity of SOD2 to signal activation and maintain mitochondrial redox balance. We demonstrate that Akt1 binds to and phosphorylates the C terminus of Hsp70 on Serine631, which inhibits CHIP-mediated SOD2 degradation thereby stabilizing and promoting SOD2 import. Conversely, increased mitochondrial-H2O2 formation disrupts Akt1-mediated phosphorylation of Hsp70, and non-phosphorylatable Hsp70 mutants decrease SOD2 import, resulting in mitochondrial oxidative stress. Our findings identify Hsp70 phosphorylation as a physiological mechanism essential for regulation of mitochondrial redox balance.

Persistent pulmonary hypertension alters the epigenetic characteristics of endothelial nitric oxide synthase gene in pulmonary artery endothelial cells in a fetal lamb model.

Decreased expression of endothelial nitric oxide synthase (eNOS), a key mediator of perinatal transition, characterizes persistent pulmonary hypertension of the newborn (PPHN) in neonates and a fetal lamb model; the mechanisms are unclear. We investigated whether increased DNA CpG methylation at the eNOS promoter in estrogen response elements (EREs) and altered histone code together contribute to decreased eNOS expression in PPHN. We isolated pulmonary artery endothelial cells (PAEC) from fetal lambs with PPHN induced by prenatal ductus arteriosus constriction from 128 to 136 days gestation or gestation-matched twin controls. We measured right ventricular systolic pressure (RVSP) and Fulton index and determined eNOS expression in PAEC in control and PPHN lambs. We determined DNA CpG methylation by pyrosequencing and activity of ten eleven translocase demethylases (TET) by colorimetric assay. We quantified the occupancy of transcription factors, specificity protein 1 (Sp1), and estrogen receptors and density of four histone marks around Sp1 binding sites by chromatin immunoprecipitation (ChIP) assays. Fetal lambs with PPHN developed increased RVSP and Fulton index. Levels of eNOS mRNA and protein were decreased in PAEC from PPHN lambs. PPHN significantly increased the DNA CpG methylation in eNOS promoter and decreased TET activity in PAEC. PPHN decreased Sp1 occupancy and density of the active mark, lysine 12 acetylation of histone 4, and increased density of the repression mark, lysine 9 trimethylation of histone 3 around Sp1 binding sites in eNOS promoter. These results suggest that epigenetic modifications are primed to decrease Sp1 binding at the eNOS gene promoter in PPHN.

Effects of neonatal hyperoxia on the critical period of postnatal development of neurochemical expressions in brain stem respiratory-related nuclei in the rat.

We have identified a critical period of respiratory development in rats at postnatal days P12-13, when inhibitory influence dominates and when the response to hypoxia is at its weakest. This critical period has significant implications for Sudden Infant Death Syndrome (SIDS), the cause of which remains elusive. One of the known risk factors for SIDS is prematurity. A common intervention used in premature infants is hyperoxic therapy, which, if prolonged, can alter the ventilatory response to hypoxia and induce sustained inhibition of lung alveolar growth and pulmonary remodeling. The goal of this study was to test our hypothesis that neonatal hyperoxia from postnatal day (P) 0 to P10 in rat pups perturbs the critical period by altering the normal progression of neurochemical development in brain stem respiratory-related nuclei. An in-depth, semiquantitative immunohistochemical study was undertaken at P10 (immediately after hyperoxia and before the critical period), P12 (during the critical period), P14 (immediately after the critical period), and P17 (a week after the cessation of hyperoxia). In agreement with our previous findings, levels of cytochrome oxidase, brain-derived neurotrophic factor (BDNF), TrkB (BDNF receptor), and several serotonergic proteins (5-HT1A and 2A receptors, 5-HT synthesizing enzyme tryptophan hydroxylase [TPH], and serotonin transporter [SERT]) all fell in several brain stem respiratory-related nuclei during the critical period (P12) in control animals. However, in hyperoxic animals, these neurochemicals exhibited a significant fall at P14 instead. Thus, neonatal hyperoxia delayed but did not eliminate the critical period of postnatal development in multiple brain stem respiratory-related nuclei, with little effect on the nonrespiratory cuneate nucleus.

Attenuation of endoplasmic reticulum stress by caffeine ameliorates hyperoxia-induced lung injury.

Rodent pups exposed to hyperoxia develop lung changes similar to bronchopulmonary dysplasia (BPD) in extremely premature infants. Oxidative stress from hyperoxia can injure developing lungs through endoplasmic reticulum (ER) stress. Early caffeine treatment decreases the rate of BPD, but the mechanisms remain unclear. We hypothesized that caffeine attenuates hyperoxia-induced lung injury through its chemical chaperone property. Sprague-Dawley rat pups were raised either in 90 (hyperoxia) or 21% (normoxia) oxygen from postnatal day 1 (P1) to postnatal day 10 (P10) and then recovered in 21% oxygen until P21. Caffeine (20 mg/kg) or normal saline (control) was administered intraperitoneally daily starting from P2. Lungs were inflation-fixed for histology or snap-frozen for immunoblots. Blood caffeine levels were measured in treated pups at euthanasia and were found to be 18.4 ± 4.9 µg/ml. Hyperoxia impaired alveolar formation and increased ER stress markers and downstream effectors; caffeine treatment attenuated these changes at P10. Caffeine also attenuated the hyperoxia-induced activation of cyclooxygenase-2 and markers of apoptosis. In conclusion, hyperoxia-induced alveolar growth impairment is mediated, in part, by ER stress. Early caffeine treatment protects developing lungs from hyperoxia-induced injury by attenuating ER stress.

Domain Mapping of Heat Shock Protein 70 Reveals That Glutamic Acid 446 and Arginine 447 Are Critical for Regulating Superoxide Dismutase 2 Function.

Stress-inducible heat shock protein 70 (hsp70) interacts with superoxide dismutase 2 (SOD2) in the cytosol after synthesis to transfer the enzyme to the mitochondria for subsequent activation. However, the structural basis for this interaction remains to be defined. To map the SOD2-binding site in hsp70, mutants of hsp70 were made and tested for their ability to bind SOD2. These studies showed that SOD2 binds in the amino acid 393-537 region of the chaperone. To map the hsp70-binding site in SOD2, we used a series of pulldown assays and showed that hsp70 binds to the amino-terminal domain of SOD2. To better define the binding site, we used a series of decoy peptides derived from the primary amino acid sequence in the SOD2-binding site in hsp70. This study shows that SOD2 specifically binds to hsp70 at 445GERAMT450 Small peptides containing GERAMT inhibited the transfer of SOD2 to the mitochondria and decreased SOD2 activity in vitro and in vivo To determine the amino acid residues in hsp70 that are critical for SOD2 interactions, we substituted each amino acid residue for alanine or more conservative residues, glutamine or asparagine, in the GERAMT-binding site. Substitutions of E446A/Q and R447A/Q inhibited the ability of the GERAMT peptide to bind SOD2 and preserved SOD2 function more than other substitutions. Together, these findings indicate that the GERAMT sequence is critical for hsp70-mediated regulation of SOD2 and that Glu446 and Arg447 cooperate with other amino acid residues in the GERAMT-binding site for proper chaperone-dependent regulation of SOD2 antioxidant function.

Decreased endothelial nitric oxide synthase expression and function contribute to impaired mitochondrial biogenesis and oxidative stress in fetal lambs with persistent pulmonary hypertension.

Impaired vasodilation in persistent pulmonary hypertension of the newborn (PPHN) is characterized by mitochondrial dysfunction. We investigated the hypothesis that a decreased endothelial nitric oxide synthase level leads to impaired mitochondrial biogenesis and function in a lamb model of PPHN induced by prenatal ductus arteriosus constriction. We ventilated PPHN lambs with 100% O2 alone or with inhaled nitric oxide (iNO). We treated pulmonary artery endothelial cells (PAECs) from normal and PPHN lambs with detaNONOate, an NO donor. We observed decreased mitochondrial (mt) DNA copy number, electron transport chain (ETC) complex subunit levels, and ATP levels in PAECs and lung tissue of PPHN fetal lambs at baseline compared with gestation matched controls. Phosphorylation of AMP-activated kinase (AMPK) and levels of peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) and sirtuin-1, which facilitate mitochondrial biogenesis, were decreased in PPHN. Ventilation with 100% O2 was associated with larger decreases in ETC subunits in the lungs of PPHN lambs compared with unventilated PPHN lambs. iNO administration, which facilitated weaning of FiO2 , partly restored mtDNA copy number, ETC subunit levels, and ATP levels. DetaNONOate increased eNOS phosphorylation and its interaction with heat shock protein 90 (HSP90); increased levels of superoxide dismutase 2 (SOD2) mRNA, protein, and activity; and decreased the mitochondrial superoxide levels in PPHN-PAECs. Knockdown of eNOS decreased ETC protein levels in control PAECs. We conclude that ventilation with 100% O2 amplifies oxidative stress and mitochondrial dysfunction in PPHN, which are partly improved by iNO and weaning of oxygen.

Efficient transgenic rat production by a lentiviral vector.

A lentiviral construct for an enhanced green fluorescent protein (eGFP) driven by a chicken beta-actin promoter, cytomegalovirus enhancer, and intronic sequences from rabbit beta-globin (CAG) was used to produce transgenic lines of rats for evaluation of the usefulness of this approach in gene function studies. Fertilized eggs were collected from inbred Dahl S and outbred Sprague-Dawley rats, and approximately 100 pl of concentrated virus were microinjected into the perivitrelline space of one-cell embryos. Of 121 embryos injected, 60 pups (49.6%) were born. Transgenic rates averaged 22% in Dahl S and 14% in Sprague-Dawley rats. Copy number ranged from one to four in the founders, and the inheritance of the transgene in a subsequent F(1) population was 48.2%. The small number of insertion sites enabled us to derive inbred transgenic lines with a single copy of the transgene within one generation. Sequencing of each transgene insertion site revealed that they inserted as single copies with a preference for the introns of genes. The CAG promoter drove high levels of eGFP expression in brain, kidney, heart, and vasculature, making it very suitable for exploring the cardiovascular function of newly discovered genes. The pattern of eGFP expression was similar across five different F(1) transgenic lines, indicating that the expression of the transgene was independent of its chromosomal position. Thus lentiviral transgenesis provides a powerful tool for the production of transgenic inbred rats and will enhance the usefulness of this species in gene discovery and target validation studies.

Central neuropeptide Y signaling ameliorates N(omega)-nitro-L-arginine methyl ester hypertension in the rat through a Y1 receptor mechanism.

Neuropeptide Y is a potent inhibitory neurotransmitter expressed in the central neurons that control blood pressure. NO also serves as an inhibitory neurotransmitter, and its deficit causes sympathetic overactivity, which then contributes to hypertension. This study tested the hypothesis that neuropeptide Y functions as a central neurotransmitter to lower blood pressure, therefore its increased signaling ameliorates hypertension induced by NO deficiency. Conscious neuropeptide Y transgenic male rats, overexpressing the peptide under its natural promoter, and nontransgenic littermates (controls) were used in this study. Neuropeptide Y, Y1 receptor antagonist BIBP3226, or vehicle (saline) were administered continuously for 14 days into the cerebral lateral ventricle in unrestrained animals using osmotic pumps. Blood pressure was measured by radiotelemetry. Compared with control animals, transgenic overexpression of neuropeptide Y significantly ameliorated (by 9.7+/-1.5 mm Hg) NO deficiency hypertension (induced by administration of N(omega)-nitro-L-arginine methyl ester in the drinking water). This hypotensive effect of neuropeptide Y upregulation was associated with reduced proteinuria and cardiac hypertrophy and fibrosis. Central administration of neuropeptide Y in nontransgenic rats also reduced (by 10.2+/-1.6 mm Hg) the NO deficiency hypertension, whereas a neuropeptide Y1 receptor antagonist centrally administered in the transgenic subjects during NO deficiency hypertension completely attenuated the depressor effect of neuropeptide Y upregulation. Thus, acting at the level of the central nervous system distinctively via a Y1 receptor-mediated mechanism, endogenous neuropeptide Y exerted a potent antihypertensive function, and its enhanced signaling ameliorated NO deficiency hypertension.

Transgenic rescue demonstrates involvement of the Ian5 gene in T cell development in the rat.

A single point mutation in a novel immune-associated nucleotide gene 5 (Ian5) coincides with severe T cell lymphopenia in BB rats. We used a transgenic rescue approach in lymphopenic BB-derived congenic F344.lyp/lyp rats to determine whether this mutation is responsible for lymphopenia and to establish the functional importance of this novel gene. A 150-kb P1 artificial chromosome (PAC) transgene harboring a wild-type allele of the rat Ian5 gene restored Ian5 transcript and protein levels, completely rescuing the T cell lymphopenia in the F344.lyp/lyp rats. This successful complementation provides direct functional evidence that the Ian5 gene product is essential for maintaining normal T cell levels. It also demonstrates that transgenic rescue in the rat is a practical and definitive method for revealing the function of a novel gene.

Neuropeptide Y induces ischemic angiogenesis and restores function of ischemic skeletal muscles.

Previously we showed that neuropeptide Y (NPY), a sympathetic vasoconstrictor neurotransmitter, stimulates endothelial cell migration, proliferation, and differentiation in vitro. Here, we report on NPY's actions, receptors, and mediators in ischemic angiogenesis. In rats, hindlimb ischemia stimulates sympathetic NPY release (attenuated by lumbar sympathectomy) and upregulates NPY-Y2 (Y2) receptor and a peptidase forming Y2/Y5-selective agonist. Exogenous NPY at physiological concentrations also induces Y5 receptor, stimulates neovascularization, and restores ischemic muscle blood flow and performance. NPY-mediated ischemic angiogenesis is not prevented by a selective Y1 receptor antagonist but is reduced in Y2(-/-) mice. Nonischemic muscle vascularity is also lower in Y2(-/-) mice, whereas it is increased in NPY-overexpressing rats compared with their WT controls. Ex vivo, NPY-induced aortic sprouting is markedly reduced in Y2(-/-) aortas and spontaneous sprouting is severely impaired in NPY(-/-) mice. NPY-mediated aortic sprouting, but not cell migration/proliferation, is blocked by an antifetal liver kinase 1 antibody and abolished in mice null for eNOS. Thus, NPY mediates neurogenic ischemic angiogenesis at physiological concentrations by activating Y2/Y5 receptors and eNOS, in part due to release of VEGF. NPY's effectiveness in revascularization and restoring function of ischemic tissue suggests its therapeutic potential in ischemic conditions.

Hypotension and reduced catecholamines in neuropeptide Y transgenic rats.

The neurons that control blood pressure express neuropeptide Y. Administered centrally, this neuropeptide reduces blood pressure and anxiety, together with lowering sympathetic outflow. The generation of neuropeptide Y transgenic rats overexpressing this peptide, under its natural promoter, has allowed us to examine the role of endogenous neuropeptide Y in the long-term control of blood pressure by the sympathetic nervous system. This study tested a hypothesis that endogenous neuropeptide Y acts to reduce blood pressure and catecholamine release. Blood pressure was measured by radiotelemetry in conscious male transgenic and nontransgenic littermates (control). Novel cage with cold water and forced swimming were used as stressors. Catecholamines were determined in 24-hour urine (baseline) and plasma (cold water stress) by a radioenzymatic assay. Blood pressures in baseline and during the stresses were significantly reduced in the transgenic rats. The lower blood pressure was associated with reduced catecholamines, lower decrease in pressure after autonomic ganglionic blockade, and increased longevity. Data obtained through the use of this transgenic rat model support and extend the evidence for the previously postulated sympatholytic and hypotensive effects of neuropeptide Y and provide novel evidence for an important physiological role of endogenous peptide in blood pressure regulation. As indicated by the increased longevity of these rats, in long-term regulation, these buffering actions of neuropeptide Y may have important cardiovascular protective effects against sympathetic hyperexcitation.