Mieczyslaw Michalkiewicz

Knockout Rats via Embryo Microinjection of Zinc-Finger Nucleases

Targeted gene disruption in rats paves the way for new human disease models. The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. By using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or messenger RNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25 to 100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within 4 months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.

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 NPYs 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. NPYs 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

Abstract—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.

Central Neuropeptide Y Signaling Ameliorates N(ω)-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(&ohgr;)-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.

Sympathetic thyroidal vasoconstriction is not blocked by a neuropeptide Y antagonist or antiserum

Sympathetic nerve fibers to thyroid blood vessels contain both norepinephrine (NE) and neuropeptide Y (NPY). To assess the involvement of endogenous NPY in the sympathetic neural control of thyroid blood flow, appropriate doses of a selective NPY antagonist, alpha-trinositol, and an NPY antiserum (NPY-AS) were used during cervical sympathetic trunk stimulation in anesthetized rats. During all experiments, thyroid blood flow was continuously monitored by laser Doppler blood flowmetry. Neither alpha-trinositol nor NPY-AS blocked the thyroidal vasoconstriction evoked by either the first or second stimulation of the cervical sympathetic trunks. Our results suggest that NPY is not involved either directly or indirectly during acute sympathetic vasoconstriction in the rat thyroid gland.

Effect of peripheral axotomy on pain-related behavior and dorsal root ganglion neurons excitability in NPY transgenic rats

In order to clarify the physiologic role of NPY in sensory processing, we obtained intracellular recordings of DRG neurons from wild type (WT) and NPY overexpressing transgenic rats (NPY-TG) before and after injury. We investigated medium and large diameter DRG neurons since upregulation of NPY peptide following the nerve injury occurs primarily in those cells. Neurons were classified as Aalpha/beta and Adelta using conduction velocity and action potential duration. Prior to the injury, Aalpha/beta neurons of NPY-TG rats conducted more slowly and had a more brief AHP than similar cells from the WT group. Adelta neurons at baseline conducted faster in TG animals compared to WT. Ligation of the 5th lumbar spinal nerve (SNL) produced certain changes in Aalpha/beta cells that were evident only in the TG group. These include increased refractory period, increased input resistance, AHP prolongation and a depolarizing shift in threshold for AP initiation. The expected injury-induced CV slowing was not seen in NPY-TG Aalpha/beta cells. In the Adelta cell group, injury produced a depolarizing shift in the resting membrane potential, an increase in AP duration and decrease in AHP and refractory period duration only in WT rats, while NPY-TG cells lacked these injury-induced changes. Behavior tests showed diminished sensory response to nerve injury in NPY-TG rats, i.e., shorter duration of enhanced pain-related behavior and attenuation of contralateral effect. In conclusion, our observations suggest that NPY overexpression leads to reduced neuronal activity following nerve injury in a cell-specific manner.

Helodermin, but not cholecystokinin, somatostatin, or thyrotropin releasing hormone, acutely increases thyroid blood flow in the rat.

In the present study, we investigated whether peptides located within the thyroid gland, but not directly found in nerve fibers associated with blood vessels, might influence thyroid blood flow. Specifically, we evaluated the effects of helodermin, cholecystokinin (CCK), somatostatin (SRIF) and thyrotropin releasing hormone (TRH) given systemically on thyroid blood flow and circulating thyroid hormone levels. Blood flows in the thyroid and six other organs were measured in male rats using 141Ce-labeled microspheres. Circulating thyrotropin (TSH) and thyroid hormone levels were monitored by RIA. Helodermin (10(-10) mol/100 g BW, i.v. over 4 min) markedly elevated thyroid blood flow (52 +/- 6 vs. 10 +/- 2 ml/min.g in vehicle-infused rats; n = 5). Blood flows to the salivary gland, pancreas, lacrimal gland and stomach (but not adrenal and kidney) were also increased during helodermin infusions. CCK, SRIF, and TRH were without effect on blood flows to the thyroid and other organs even though these peptides were tested at higher molar doses than helodermin. Helodermin, CCK, or SRIF did not affect thyroid hormone or plasma calcium levels. As expected however, plasma TSH and T3 levels were increased at 20 min and 2 h, respectively, following TRH infusions. Since helodermin shares sequence homology with VIP, we next compared the relative effects of these two peptides on thyroid and other organ blood flows. VIP (10(-11) mol/100 g BW, i.v.) was more potent in increasing blood flows to the thyroid, salivary gland, and pancreas than an equimolar dose of helodermin. This study shows that while helodermin, like VIP, has the ability to increase thyroid and other organ blood flows, it appears to be a less potent vasodilator.

Adenohypophyseal Vasoactive Intestinal Peptide and Neuropeptide Y Responses to Hypothyroidism Are Abolished after Anterolateral Deafferentation of the Hypothalamus

We examined whether hypothyroidism-induced increases in the anterior pituitary content of vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) are mediated by the hypothalamus. Male Sprague-Dawley rats were anesthetized and one of the following surgeries was performed: (1) sham thyroidectomy, (2) thyroidectomy, (3) thyroidectomy plus surgical anterolateral deafferentation of the medial basal hypothalamus, or (4) thyroidectomy and sham deafferentation of the hypothalamus (knife was inserted but not rotated). Two weeks after surgery, animals were killed and tissue samples collected for measurement of the anterior pituitary VIP and NPY and plasma thyroid-stimulating hormone (TSH), thyroxine, and prolactin concentrations (by RIA). Reverse-phase HPLC showed that VIP and NPY immunoreactivities in the anterior pituitary extracts are eluted in the positions identical to synthetic VIP and NPY, respectively. Only data from those animals with complete symmetric cuts located at the posterior border of the optic chiasm were included for analysis. In the thyroidectomized rats the anterior pituitary contents of VIP and NPY were significantly increased. These responses were almost completely prevented by the anterolateral deafferentation of the hypothalamus. Sham hypothalamic deafferentation had no effect on the pituitary neuropeptide responses to hypothyroidism. Anterolateral deafferentation of the hypothalamus also significantly blunted plasma TSH responses to hypothyroidism. These data suggest that some hypothalamic factor is involved in the mediation of the effect of hypothyroidism on the pituitary content of VIP and NPY.

Vasoactive Intestinal Peptide Enhances Thyroidal Iodide Uptake During Dietary Iodine Deficiency

The presence of vasoactive intestinal peptide and neuropeptide Y in thyroid nerves and their effects on thyroid blood flow are well known. However, the effects of these two neuropeptides on the various processes involved in thyroid hormone biosynthesis and release have not been fully explored. We have now tested these two peptides for effects on an early step in thyroid hormone biosynthesis, namely iodide uptake, a process which is comprised of trapping and organification. In these experiments, we have used anesthetized adult male rats pretreated with thyroxine or fed a low iodine diet to increase thyroidal sensitivity. Vasoactive intestinal peptide significantly increased iodide uptake in rats fed an iodine deficient diet but not in those fed a normal iodine diet. This effect disappeared if animals were pretreated with propylthiouracil. Neuropeptide Y did not alter iodide uptake in rats on either the low or the high iodine diet, regardless of the presence or absence of propylthiouracil. The effect of vasoactive intestinal peptide on iodide uptake could be due to its influence on the organification of iodine, or on thyroid blood flow, or on both processes.

Expression of preproNPY and precursor VIP mRNAs in rats under hypo- or hyperthyroid conditions.

Both neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) are present in thyroid nerves and have been shown to alter thyroid activity. The present study was conducted to determine whether hypo- or hyperthyroidism is associated with changes in the expression of the mRNAs for these neuropeptides in the major ganglia which supply nerves to the thyroid or within the thyroid gland itself. Hypo- or hyperthyroid conditions were induced by the administration of propylthiouracil (PTU) or thyroxine (T(4)), respectively, for 6 days. Control rats received vehicle injections. Total RNA from superior cervical ganglia (SCG), local thyroid ganglia, thyroid gland, and selected other tissues was extracted and mRNA levels were analyzed using Northern blot procedures. No significant changes in preproNPY or precursor VIP mRNA levels were detected in the SCG or the local thyroid ganglia in response to PTU or T(4) treatment. However, PTU treatment was associated with an increase in preproNPY mRNA levels in the thyroid gland itself. These results indicate that changes within the thyroid axis in response to these hypo- and hyperthyroid conditions do not include alterations in steady-state preproNPY or precursor VIP mRNA concentrations in the major ganglia which supply nerves to the thyroid gland. However, intrathyroidal preproNPY mRNA levels are increased as a consequence of the thyroidal adaptation to a PTU challenge.