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A 39-year-old previously healthy man presented with insidiously progressive paresthesia in his lower extremities and worsening of gait and balance. MRI demonstrated T2-hyperintense signal abnormalities involving the thalami, midbrain, dentate nuclei, and adjacent deep cerebellar white matter, which are characteristic of cerebrotendinous xanthomatosis (CTX) (figure). Magnetic resonance spectroscopy revealed typical lipid peaks at 0.9 and 1.3 ppm,1 and serology analysis indicated sterol 27-hydroxylase deficiency,2 confirming the diagnosis. CTX is a rare lipid storage disease with unique clinical and imaging findings, and effective treatment with positive outcomes if initiated early, raising the importance of awareness and early recognition of this disease.2

Objective: The alpha-synuclein gene (SNCA) is implicated in both Parkinson's disease (PD) and dementia with Lewy bodies (DLB). The purpose of this case study was to describe the neuropsychological profile, clinical trajectory, and treatment course of an individual with a known SNCA gene duplication who was followed over the course of three years. Methods: The patient was a healthy man who developed olfactory changes in early adulthood followed by parkinsonism and cognitive concerns around age 40. He underwent serial neurologic and neuropsychological evaluations and neuroimaging, as well as genetic testing for PD gene mutations. He consented to share his medical information to increase awareness of his condition. Results: Initial neuropsychological evaluation (age 44) revealed mild cognitive impairment primarily affecting executive and frontal/subcortical functions. Follow-up evaluations showed rapid cognitive decline that far surpassed the patient's Parkinsonism, which responded well to carbidopa-levodopa. As symptoms progressed, he also developed features characteristic of DLB, including cognitive fluctuations, rapid eye movement sleep behavior disorder, and visual hallucinations. Conclusion: SNCA gene duplication has classically been associated with a slowly progressive syndrome closely resembling idiopathic PD, but less frequently it can cause rapidly progressive dementia. This case study is the first to describe this rare phenotype in terms of its full neuropsychological profile and trajectory. The case highlights the value of a transdisciplinary evaluation and treatment and brings up important ethical and practical issues that should be considered when working with patients who have suspected or known genetic disorders.

Primary lateral sclerosis (PLS) is a neurodegenerative disease characterized by progressive upper motor neuron dysfunction. Because PLS patients represent only 1 to 4% of patients with adult motor neuron diseases, there is limited information about the disease's natural history. The objective of this study was to establish a large multicenter retrospective longitudinal registry of PLS patients seen at Northeast ALS Consortium (NEALS) sites to better characterize the natural progression of PLS.

Introduction/Aims. Primary lateral sclerosis (PLS) is exceedingly rare and has been an enigmatic disease. Recent progress has drastically changed this perception, with early biomarkers being investigated and potential medications for PLS emerging at the preclinical stage. The aim of this paper is to describe a study of PLS natural history and discuss the limitations and proposed solutions to the study of a rare and slowly progressive disease. Methods. The PLS Natural History Study is a 30-site, 24-month, prospective study that is supported by multiple funding sources. The study aims to enroll 50 early PLS (disease duration ≤4 years) and 50 definite PLS (disease duration 4 to 15 years) participants using modified PLS Diagnostic Criteria. Smartphone-based assessments including semi-quantitative and quantitative measures and patient-reported outcomes are utilized. In-person quantitative measures are also completed during site visits. The change in the PLS Functional Rating Scale score is the primary outcome. The study utilizes the NeuroBANK® patient-centric data capture and management platform. The biostatistical analysis plan has been developed. Results. In one year, 28 participants have been recruited. Enrollment has been much slower than anticipated due to the COVID-19 pandemic, the rarity of PLS, and potential study competition for internal resources from ALS clinical trials. Discussion. We discuss the need for more innovative methods to enroll and study individuals with such rare diseases and propose a number of mechanisms by which more efficient enrollment could be facilitated.

Endothelin 1 (ET-1), mainly produced from vascular endothelial cells, induces vasoconstriction in physiological conditions. The endothelin receptor antagonist is among the most effective agents for pulmonary hypertension. However, little is known about the production source of ET-1 in inflammation and immunity. Here, we studied whether T cell-mediated ET-1 production system exists and operates independent of the production system in vascular endothelial cells. ET-1 production was readily detectable in the culture supernatant of human PBMCs and murine spleen cells stimulated with anti-CD3 antibody. Immunocytostaining showed that ET-1-producing cells emerged only in PBMCs stimulated with anti-CD3 antibody. Using the Transwell system, both murine and human monocytes sorted with magnetic beads in the inner chamber produced ET-1 when T cells were activated with antigen or anti-CD3 antibody in the outer chamber. This ET-1 production was inhibited by anti-IFN-γ and/or TNF-α antibody. Furthermore, monocytes purified from ETflox/flox;Tie2-Cre( + ) mice, which conditionally lack ET-1 in hematopoietic stem cells and vascular endothelial cells, did not produce ET-1 even when stimulated by antigen-specific T cell activation. This study demonstrates the existence of an immune-mediated ET-1 production induced by T cells upon activation through IFN-γ and TNF-α.

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a rare white matter degenerative disease characterized by both axonal and glial injury due to a defect in the CSF1R gene. In this report, we describe ALSP in a previously healthy 40-year-old woman presenting with insidiously progressive confusion, memory loss, and loss of social inhibitions. Characteristic magnetic resonance imaging findings for ALSP elucidated the diagnosis, including chronic foci of diffusion restriction in a non-vascular distribution, lack of temporal/infratentorial involvement, cortical sparing, and lack of enhancement. CSF1R genetic testing further confirmed the diagnosis and the patient underwent supportive medical management for symptom control. ALSP can pose a unique diagnostic challenge given its particular adult-onset presentation, but early recognition is key given the poor prognosis and the potential for family genetic testing.

Supplemental Digital Content is available in the text., Hereditary spastic paraplegias (HSPs) are characterized by progressive spasticity and weakness in the lower extremities that result from length-dependent central to peripheral axonal degeneration. Mutations in the non-imprinted Prader-Willi/Angelman syndrome locus 1 (NIPA1) transmembrane protein cause an autosomal dominant form of HSP (SPG6). Here, we report that transgenic (Tg) rats expressing a human NIPA1/SPG6 mutation in neurons (Thy1.2-hNIPA1G106R) show marked early onset behavioral and electrophysiologic abnormalities. Detailed morphologic analyses reveal unique histopathologic findings, including the accumulation of tubulovesicular organelles with endosomal features that start at axonal and dendritic terminals, followed by multifocal vacuolar degeneration in both the CNS and peripheral nerves. In addition, the NIPA1G106R mutation in the spinal cord from older Tg rats results in an increase in bone morphogenetic protein type II receptor expression, suggesting that its degradation is impaired. This Thy1.2-hNIPA1G106R Tg rat model may serve as a valuable tool for understanding endosomal trafficking in the pathogenesis of a subgroup of HSP with an abnormal interaction with bone morphogenetic protein type II receptor, as well as for developing potential therapeutic strategies for diseases with axonal degeneration and similar pathogenetic mechanisms.

金沢大学医薬保健研究域医学系, Orexin-A and orexin-B are hypothalamic neuropeptides that play critical roles in the maintenance of wakefulness. Intracerebroventricular (ICV) administration of orexin-A has been shown to promote wakefulness and suppress both rapid eye movement (REM) sleep and non-REM (NREM) sleep through the orexin receptor-1 (OX1R) and orexin receptor-2 (OX2R). Here, we elucidated the differential roles of orexin receptors in the regulation of sleep and wakefulness by comparing the effects of ICV orexin-A administration in wild-type, OX1R, and OX2R mice. The effects of orexin-A on wakefulness and NREM sleep were significantly attenuated in both knock-out mice as compared with wild-type mice, with substantially larger attenuation in OX2R mice than in OX1R mice. These results suggest that although the OX2R-mediated pathway has a pivotal role in the promotion of wakefulness, OX1R also plays additional roles in promoting arousal. In contrast, suppression of REM sleep by orexin-A administration was slightly and similarly attenuated in both OX1R and OX2R mice, suggesting a comparable contribution of the two receptors to REM sleep suppression. Histological studies demonstrated differential distributions of each receptor subtype in distinct neuronal populations with specific neurotransmitter identities in brainstem cholinergic/monoaminergic neurons. In the laterodorsal tegmental and pedunculopontine tegmental nuclei especially, cholinergic neurons exclusively expressed OX1R mRNA, but OX2R mRNA was expressed mainly in GABAergic putative interneurons. Thus, each orexin receptor subtype plays differential roles in gating NREM and REM sleep through distinct neuronal pathways. © 2011 by the authors.

Endothelin (ET) 1 is a potent vasoconstrictor peptide produced by vascular endothelial cells and implicated in various pathophysiologic states involving abnormal vascular tone. Homozygous ET-1 null mice have craniofacial and cardiac malformations that lead to neonatal death. To study the role of ET-1 in adult vascular physiology, we generated a mouse strain (ET-1 flox/flox ;Tie2-Cre mice) in which ET-1 is disrupted specifically in endothelial cells. ET-1 peptide levels in plasma, heart, lung, kidney, and brain homogenates were reduced by 65% to 80% in these mice. mRNA levels for ET receptors were unaltered except that the ET A receptor mRNA was upregulated in the heart. ET-1 flox/flox ;Tie2-Cre mice had mean blood pressures 10 to 12 mm Hg lower than genetic controls. In contrast, the blood pressure of mice systemically heterozygous for the ET-1 null allele (ET-1 dlox/+ mice) was unchanged compared with wild-type littermates. Despite the lower basal blood pressure, acute pharmacological responses to angiotensin II, captopril, phenylephrine, bradykinin, N G -nitro- l -arginine methyl ester, and exogenous ET-1 were normal in ET-1 flox/flox ;Tie2-Cre mice. These results support an essential role of endothelial-derived ET-1 in the maintenance of basal vascular tone and blood pressure. Normal pharmacological responses of ET-1 flox/flox ;Tie2-Cre mice suggest that the renin-angiotensin system, the adrenergic system, and NO are not significantly altered by endothelial ET-1. Taken in conjunction with other lines of genetically altered mice, our results provide evidence for a paracrine vasoregulatory pathway mediated by endothelial cell–derived ET-1 acting on the vascular smooth muscle ET A receptor.

Pharmacological studies of narcoleptic canines indicate that exaggerated pontine cholinergic transmission promotes cataplexy. As disruption of orexin (hypocretin) signaling is a primary defect in narcolepsy with cataplexy, we investigated whether markers of cholinergic synaptic transmission might be altered in mice constitutively lacking orexin receptors (double receptor knockout; DKO). mRNA for Choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and the high-affinity choline transporter (CHT1) but not acetylcholinesterase (AChE) was significantly higher in samples from DKO than wild-type (WT) mice. This was region-specific; levels were elevated in samples from the laterodorsal tegmental nucleus (LDT) and the fifth motor nucleus (Mo5) but not in whole brainstem samples. Consistent with region-specific changes, we were unable to detect significant differences in Western blots for ChAT and CHT1 in isolates from brainstem, thalamus and cortex or in ChAT enzymatic activity in the pons. However, using ChAT immunocytochemistry, we found that while the number of cholinergic neurons in the LDT and Mo5 were not different, the intensity of somatic ChAT immunostaining was significantly greater in the LDT, but not Mo5, from DKO than from WT mice. We also found that ChAT activity was significantly reduced in cortical samples from DKO compared with WT mice. Collectively, these findings suggest that the orexins can regulate neurotransmitter expression and that the constitutive absence of orexin signaling results in an up-regulation of the machinery necessary for cholinergic neurotransmission in a mesopontine population of neurons that have been associated with both normal rapid eye movement sleep and cataplexy.

Aims Although endothelin-1 (ET-1) has been suggested to contribute to the pathogenesis of neointima formation and atherosclerosis, the individual roles of ET-1 derived from certain cell types in this disease remain unclear. In this study, we determined the role of vascular endothelial ET-1 on vascular inflammation and neointima formation using vascular endothelial ET-1-knockout [ET-1f/f; Tie2-Cre (+)] mice. Methods and results Intimal hyperplasia was induced by complete ligation of the left carotid artery in 12-week-old male ET-1f/f;Tie2-Cre (+) mice ( n = 35) and the wild-type (WT) littermates ( n = 34). Following this intervention, neointima formation was reduced in ET-1f/f;Tie2-Cre (+) mice compared with the WT mice, independent of the difference in blood pressure. This reduction was associated with a decrease in inflammatory cell recruitment to the vessel wall, which was accompanied by reduced expression levels of endothelial adhesion molecules as well as chemokines and a decrease in vascular smooth muscle cell proliferation. Conclusion The results of our study provide direct evidence for the role of vascular endothelial ET-1 in mediating vascular inflammation and neointima formation following vascular injury in addition to promoting vasoconstriction and cell proliferation. Furthermore, this study suggests a strategy for the efficient design of ET receptor antagonists with targeted inhibition of ET-1 signalling in vascular endothelial cells.

Endothelin 1 (ET-1), a potent vasoconstrictor peptide expressed by endothelium, is also produced in the heart in response to a variety of stresses. It induces hypertrophy in cultured cardiac myocytes but only at concentrations far greater than those found in plasma. We tested whether ET-1 generated by cardiac myocytes in vivo is a local signal for cardiac hypertrophy. To avoid the perinatal lethality seen in systemic ET-1 -null mice, we used the Cre/loxP system to generate mice with cardiac myocyte-specific disruption of the ET-1 gene. We used the α-myosin heavy chain promoter to drive expression of Cre and were able to obtain 75% reduction in ET-1 mRNA in cardiac myocytes isolated from these mice at baseline and after stimulation, in vivo , for 24 h with tri-iodothyronine (T3). Necropsy measurements of cardiac mass indexed for body weight showed a 57% reduction in cardiac hypertrophy in response to 16 days of exogenous T3 in mice homozygous for the disrupted ET-1 allele compared to siblings with an intact ET-1 gene. Moreover, in vivo MRI showed only a 3% increase in left ventricular mass indexed for body weight in mice with the disrupted allele after 3 weeks of T3 treatment versus a 27% increase in mice with an intact ET-1 gene. A reduced hypertrophic response was confirmed by planimetry of cardiac myocytes. We conclude that ET-1, produced locally by cardiac myocytes, and acting in a paracrine/autocrine manner, is an important signal for myocardial hypertrophy that facilitates the response to thyroid hormone.

Narcolepsy-cataplexy, a neurological disorder associated with the absence of hypothalamic orexin (hypocretin) neuropeptides, consists of two underlying problems: inability to maintain wakefulness and intrusion of rapid eye movement (REM) sleep into wakefulness. Here we document, using behavioral, electrophysiological, and pharmacological criteria, two distinct classes of behavioral arrests exhibited by mice deficient in orexin-mediated signaling. Both OX2R(-/-) and orexin(-/-) mice are similarly affected with behaviorally abnormal attacks of non-REM sleep ("sleep attacks") and show similar degrees of disrupted wakefulness. In contrast, OX2R(-/-) mice are only mildly affected with cataplexy-like attacks of REM sleep, whereas orexin(-/-) mice are severely affected. Absence of OX2Rs eliminates orexin-evoked excitation of histaminergic neurons in the hypothalamus, which gate non-REM sleep onset. While normal regulation of wake/non-REM sleep transitions depends critically upon OX2R activation, the profound dysregulation of REM sleep control unique to the narcolepsy-cataplexy syndrome emerges from loss of signaling through both OX2R-dependent and OX2R-independent pathways.

Trypanosoma cruzi is the aetiological agent of Chagas' disease, an important cause of chronic cardiomyopathy. We previously demonstrated a role for endothelin-1 (ET-1) in the pathogenesis of chagasic heart disease. In order to explore further the significance of ET-1 in chagasic heart disease, we infected ET-1 (flox/flox); alpha-MHC-Cre(+) (ET-1KO) mice, in which the ET-1 gene has been deleted from cardiac myocytes, with 10(4) T. cruzi (Brazil strain) trypomastigotes. As controls, we used ET-1 (flox/flox);Cre(-) (FLOX) and C57BL/6x129sv (WT) mice. All mice survived and were evaluated 150-160 days post-infection. Cardiac magnetic resonance imaging revealed a significant increase in right ventricular internal diameter in all infected animals except ET-1KO mice (control WT, 1.6+/-0.10 mm; infected WT, 2.8+/-0.15 mm; control FLOX, 2.04+/-0.02 mm; infected FLOX, 2.76+/-0.28 mm). There was no significant difference in right ventricular internal diameter between infected and uninfected ET-1KO mice (control ET-1KO, 1.83+/-0.11 mm; infected ET-1KO, 2.14+/-0.20 mm). In another series of experiments, transthoracic echocardiography was performed on uninfected as well as infected ET-1KO mice, and uninfected and infected FLOX mice. Both infected groups had an increased left ventricular end-diastolic diameter and reduced fractional shortening. In addition, relative wall thickness was also decreased in infected animals. However, the magnitude of these changes was less in infected ET-1KO mice. These data provide further support for a role for ET-1 in the pathogenesis of chronic chagasic heart disease, and indicate that cardiac myocytes are an important source of ET-1 in this disease.

Although the cellular and molecular mechanisms governing angiogenesis are only beginning to be understood, signaling through endothelial-restricted receptors, particularly receptor tyrosine kinases, has been shown to play a pivotal role in these events. Recent reports show that EphB receptor tyrosine kinases and their transmembrane-type ephrin-B2 ligands play essential roles in the embryonic vasculature. These studies suggest that cell-to-cell repellent effects due to bidirectional EphB/ephrin-B2 signaling may be crucial for vascular development, similar to the mechanism described for neuronal development. To test this hypothesis, we disrupted the precise expression pattern of EphB/ephrin-B2 in vivo by generating transgenic (CAGp-ephrin-B2 Tg) mice that express ephrin-B2 under the control of a ubiquitous and constitutive promoter, CMV enhancer-β-actin promoter-β-globin splicing acceptor (CAG). These mice displayed an abnormal segmental arrangement of intersomitic vessels, while such anomalies were not observed in Tie-2p-ephrin-B2 Tg mice in which ephrin-B2 was overexpressed in only vascular endothelial cells (ECs). This finding suggests that non-ECs expressing ephrin-B2 alter the migration of ECs expressing EphB receptors into the intersomitic region where ephrin-B2 expression is normally absent. CAGp-ephrin-B2 Tg mice show sudden death at neonatal stages from aortic dissecting aneurysms due to defective recruitment of vascular smooth muscle cells to the ascending aorta. EphB/ephrin-B2 signaling between endothelial cells and surrounding mesenchymal cells plays an essential role in vasculogenesis, angiogenesis, and vessel maturation.

Endocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme. Here, we created Tie2-Cre transgenic mice, in which expression of Cre recombinase is driven by an endothelial-specific promoter/enhancer. To analyze the lineage of Cre expressing cells, we used CAG-CAT-Z transgenic mice, in which expression of lacZ is activated only after Cre-mediated recombination. We detected pan-endothelial expression of the Cre transgene in Tie2-Cre;CAG-CAT-Z double-transgenic mice. This expression pattern is almost identical to Tie2-lacZ transgenic mice. However, interestingly, we observed strong and uniform lacZ expression in mesenchymal cells of the atrioventricular canal of Tie2-Cre;CAG-CAT-Z double-transgenic mice. We also detected lacZ expression in the mesenchymal cells in part of the proximal cardiac outflow tract, but not in the mesenchymal cells of the distal outflow tract and branchial arch arteries. LacZ staining in Tie2-Cre;CAG-CAT-Z embryos is consistent with endocardial–mesenchymal transformation in the atrioventricular canal and outflow tract regions. Our observations are consistent with previously reported results from Cx43-lacZ, Wnt1-Cre;R26R, and Pax3-Cre;R26R transgenic mice, in which lacZ expression in the cardiac outflow tract identified contributions in part from the cardiac neural crest. Tie2-Cre transgenic mice are a new genetic tool for the analyses of endothelial cell-lineage and endothelial cell–specific gene targeting.

Neurons containing the neuropeptide orexin (hypocretin) are located exclusively in the lateral hypothalamus and send axons to numerous regions throughout the central nervous system, including the major nuclei implicated in sleep regulation. Here, we report that, by behavioral and electroencephalographic criteria, orexin knockout mice exhibit a phenotype strikingly similar to human narcolepsy patients, as well as canarc-1 mutant dogs, the only known monogenic model of narcolepsy. Moreover, modafinil, an anti-narcoleptic drug with ill-defined mechanisms of action, activates orexin-containing neurons. We propose that orexin regulates sleep/wakefulness states, and that orexin knockout mice are a model of human narcolepsy, a disorder characterized primarily by rapid eye movement (REM) sleep dysregulation.

Objective To describe the clinical, radiological, and histopathological features of a fatal case of progressive multifocal leukoencephalopathy (PML) in a patient with multiple sclerosis treated with natalizumab. We will use this case to review PML risk stratification and diagnosis. Design Case report. Setting Tertiary referral center hospitalized care. Patient A 55-year-old, JC virus (JCV) antibody–positive patient with multiple sclerosis who died of PML after receiving 45 infusions of natalizumab. Main Outcome Measures Brain magnetic resonance imaging and cerebrospinal fluid JCV DNA polymerase chain reaction results. Results The patient developed subacute onset of bilateral blindness following his 44th dose of natalizumab. Ophthalmologic examination was normal, the brain magnetic resonance imaging was not suggestive of PML, and cerebrospinal fluid analysis did not reveal the presence of JCV DNA. The patient was subsequently treated for a presumed multiple sclerosis relapse with high-dose corticosteroids. Two weeks after his 45th dose of natalizumab, he developed hemiplegia that evolved into quadriparesis. Repeated magnetic resonance imaging and cerebrospinal fluid studies were diagnostic for PML. Postmortem histopathological analysis demonstrated PML-associated white matter and cortical demyelination. Conclusions The risks and benefits of natalizumab must be reassessed with continued therapy duration. When there is high clinical suspicion for PML in the setting of negative test results, close clinical vigilance is indicated, natalizumab treatment should be suspended, and JCV polymerase chain reaction testing and brain magnetic resonance imaging scans should be repeated.

To investigate how cholinergic systems regulate aspects of the sleep disorder narcolepsy, we video-monitored mice lacking both orexin (hypocretin) receptors (double knockout; DKO mice) while pharmacologically altering cholinergic transmission. Spontaneous behavioral arrests in DKO mice were highly similar to those reported in orexin-deficient mice and were never observed in wild-type (WT) mice. A survival analysis revealed that arrest lifetimes were exponentially distributed indicating that random, Markovian processes determine arrest lifetime. Low doses (0.01, 0.03 mg/kg, i.p.), but not a high dose (0.08 mg/kg, i.p.) of the cholinesterase inhibitor physostigmine increased the number of arrests but did not alter arrest lifetimes. The muscarinic antagonist atropine (0.5 mg/kg, i.p.) decreased the number of arrests, also without altering arrest lifetimes. To determine if muscarinic transmission in pontine areas linked to REM sleep control also influences behavioral arrests, we microinjected neostigmine (50 nl, 62.5 µM) or neostigmine + atropine (62.5 µM and 111 µM respectively) into the nucleus pontis oralis and caudalis. Neostigmine increased the number of arrests in DKO mice without altering arrest lifetimes but did not provoke arrests in WT mice. Co-injection of atropine abolished this effect. Collectively, our findings establish that behavioral arrests in DKO mice are similar to those in orexin deficient mice and that arrests have exponentially distributed lifetimes. We also show, for the first time in a rodent narcolepsy model, that cholinergic systems can regulate arrest dynamics. Since perturbations of muscarinic transmission altered arrest frequency but not lifetime, our findings suggest cholinergic systems influence arrest initiation without influencing circuits that determine arrest duration.

Endothelium plays important role in protective mechanism of vascular wall. The balance between endothelin-1 (ET-1) and nitric oxide provide endothelial barrier to lipoprotein retain and macrophage recruitment. In contrasts, ET-1 is also a strong vasoconstrictor. In this study, we aim to determine the role of vascular endothelial cells-derived ET-1 in the development of atherosclerosis. For that purpose, we crossbred Vascular Endothelial Cells-specific ET-1 Knockout (VEETKO) mice to ApoEKO mice. ApoE/VEET-DKO exhibited significantly lower ET-1 plasma and mRNA level as compared to ApoEKO mice. No significant differences of blood pressure, plasma cholesterol or lipid profiles were observed in both mice. Surprisingly, after 8 weeks of western diet, we found that the atherosclerotic lesion was exaggerated in the aortic sinus and brachiochepalic artery of ApoE/VEET-DKO mice (n=7) as compared to those of ApoEKO mice (n=7) (ratio/vessel wall, 0.93±0.13vs.0.49±0.09, p

Complex mechanism of the chronic inflammatory process in vascular injury and atherosclerosis involves various types of cells and cytokines. Previous in vitro studies suggest that Endothelin-1 (ET-1) is produced by those cells, induces expression of several adhesion molecules and mediates the leukocyte-endothelial interaction. In the present study, we sought to determine whether local endothelial ET-1 plays a direct effect on early inflammation and neointimal formation as a response to vascular injury in vivo . Using Cre-loxP system and Tie-2 Cre promoter, Vascular Endothelial ET-1 Knockout (VEETKO) mice was generated. The mice showed 60% reduction of ET-1 mRNA level at major organs and undetected serum ET-1 level. To induce vascular inflammation, complete ligation of the left common carotid artery was performed in 12-weeks-old male VEETKO (n=35) and Wild Type (WT) littermates (n=34). Inhibition of vascular endothelial ET-1 prevented the 4-fold increase in ET-1 mRNA level of carotid artery after ligation, in contrast to that of WT mice. This prevention resulted in lower vascular inflammation revealed by lower mRNA and protein level of PECAM-1, ICAM-1, and MCP-1 in VEETKO mice. The lack of ET-1 in endothelium further inhibited the adhesion of inflammatory cells at early days after ligation as compared to that adhered in endothelium of WT mice (cell numbers, 20.7±4.9 vs. 54.6±3.9 respectively, p

High plasma endothelin-1 (ET-1) level has been associated with diabetic cardiovascular and renal complications, which remained prevalent despite good glycemic control. Since endothelial dysfunction, which is characterized by enhanced ET-1 secretion, plays an important role in mediating diabetic complication, we hypothesized that disruption of ET-1 in endothelial cell will be protective against cardiac and renal complication of diabetes. To test this hypothesis, we injected streptozotocin to Vascular Endothelial cell Specific Endothelin-1 Knockout (VEETKO) mice, of which ET-1 expression in major organs including heart and kidney were reduced by 60%, and to their wild type (WT) littermates. Six months of diabetes increased systolic blood pressure (SBP) similarly in both genotypes, with DM-VEETKO mice have remained lower SBP than DM-WT mice (124±1.08 vs. 131.33±1.33 mmHg, p

On the basis of previous observations, endothelin 1 (ET-1) has been suggested as contributing to the pathogenesis of Chagasic cardiomyopathy. Therefore, ET-1 flox/flox ;α-MHC-Cre(+) mice in which the ET-1 gene was deleted from cardiac myocytes and ET-1 flox/flox ;Tie 2 Cre(+) mice in which the ET-1 gene was deleted from endothelial cells were infected with Trypanosoma cruzi . Genetic controls for these cell-specific ET-1 knockout mice were used. Ninety percentage of all mice survived acute infection with the Brazil strain and were evaluated 130 days postinfection. Inflammation and fibrosis were observed in all infected mice; however, fibrosis was reduced in ET-1 flox/flox ;α-MHC-Cre(+) mice. Cardiac magnetic resonance imaging revealed that infection resulted in a significant increase in right ventricular internal diameter (RVID) in all mice except ET-1 flox/flox ;α-MHC-Cre(+) mice; i.e., RVID was not changed in infected ET-1 flox/flox ;α-MHC-Cre(+) mice. Echocardiography of the left ventricle demonstrated increased left ventricular end-diastolic diameter, reduced fractional shortening, and decreased relative wall thickness in infected mice. However, the magnitude of the changes was significantly less in ET-1 flox/flox ;α-MHC-Cre(+) mice compared to other groups. These data provide further evidence of a role for ET-1, particularly cardiac myocyte-derived ET-1, in the pathogenesis of chronic Chagasic cardiomyopathy.

Endothelin 1 (ET-1) was identified as an endothelium-derived, vasoactive 21-amino-acid peptide (34). The hormone activates two G-protein-coupled receptors, endothelin A (ETA) and endothelin B (ETB), with approximately equal affinity (2, 30). Complex physiology of the endothelin system is suggested by broad tissue distribution of the ligand and its receptors. ET-1 is secreted by many cell types, including endothelial cells and cardiomyocytes (28, 34), whereas ETA and ETB receptors are expressed by cells such as vascular and nonvascular smooth muscle cells, cardiomyocytes, neurons, and renal tubular epithelial cells (2, 9, 28, 30). The circulating levels of ET-1 in plasma are 2 orders of magnitude below the concentration needed for receptor activation, suggesting that ET-1 functions locally (4). In the heart, ET-1 is produced by cardiomyocytes, fibroblasts, and endothelial cells (9, 28, 34). The cardiomyocytes predominantly express ETA receptor (9). The binding of ET-1 to both ETA and ETB receptors on cardiomyocytes results in activation of Gq signaling, increased intracellular calcium, and positive inotropy and chronotropy (5, 17, 19, 27; for review, see reference 8). In addition, ET-1 and its receptors mediate stress-induced remodeling in the mammalian heart (for a review, see reference 21). In vitro experiments show that ET-1-mediated activation of either ETA or ETB receptors on cardiomyocytes results in cellular hypertrophy (7, 18). In vivo studies demonstrate that levels of ET-1 and ETA are elevated in animal models of congestive heart failure (29, 35, 36). Providing the final proof of endothelins' molecular role in cardiac pathology, endothelin receptor antagonists inhibit the progression of cardiac remodeling in animal models of congestive heart failure (25, 28, 33). Even though pharmacological effects of endothelins are well-known, detailed tissue-specific roles of ET-1 and its receptors have never been outlined in vivo. This is in part because of neonatal lethality of systemic ETA and ET-1 knockout mice due to developmental defects in cardiac and craniofacial structures (6, 23). Therefore, utilizing the cre/loxP technology (13), we have generated mice in which the ETA gene can be deleted in a selected cell population (20). In the present study, we deleted the ETA gene in cardiomyocytes by crossing mice with loxP-flanked ETA (ETAflox) alleles with transgenic mice that express cre recombinase under the control of the cardiomyocyte-specific α-myosin heavy-chain promoter (1). The mice with cardiomyocyte-specific deletion of ETA are viable and were used to study the role of ETA in cardiac physiology and stress response to angiotensin II or isoproterenol.

Insulin receptors (IRs) on vascular endothelial cells have been suggested to participate in insulin-regulated glucose homeostasis. To directly address the role of insulin action in endothelial function, we have generated a vascular endothelial cell IR knockout (VENIRKO) mouse using the Cre-loxP system. Cultured endothelium of VENIRKO mice exhibited complete rearrangement of the IR gene and a more than 95% decrease in IR mRNA. VENIRKO mice were born at the expected Mendelian ratio, grew normally, were fertile, and exhibited normal patterns of vasculature in the retina and other tissues. Glucose homeostasis under basal condition was comparable in VENIRKO mice. Both eNOS and endothelin-1 mRNA levels, however, were reduced by approximately 30–60% in endothelial cells, aorta, and heart, while vascular EGF expression was maintained at normal levels. Arterial pressure tended to be lower in VENIRKO mice on both low- and high-salt diets, and on a low-salt diet VENIRKO mice showed insulin resistance. Thus, inactivation of the IR on endothelial cell has no major consequences on vascular development or glucose homeostasis under basal conditions, but alters expression of vasoactive mediators and may play a role in maintaining vascular tone and regulation of insulin sensitivity to dietary salt intake.

Although the cellular and molecular mechanisms governing angiogenesis are only beginning to be understood, signaling through endothelial-restricted receptors, particularly receptor tyrosine kinases, has been shown to play a pivotal role in these events. Recent reports show that EphB receptor tyrosine kinases and their transmembrane-type ephrin-B2 ligands play essential roles in the embryonic vasculature. These studies suggest that cell-to-cell repellent effects due to bidirectional EphB/ephrin-B2 signaling may be crucial for vascular development, similar to the mechanism described for neuronal development. To test this hypothesis, we disrupted the precise expression pattern of EphB/ephrin-B2 in vivo by generating transgenic (CAGp-ephrin-B2 Tg) mice that express ephrin-B2 under the control of a ubiquitous and constitutive promoter, CMV enhancer-beta-actin promoter-beta-globin splicing acceptor (CAG). These mice displayed an abnormal segmental arrangement of intersomitic vessels, while such anomalies were not observed in Tie-2p-ephrin-B2 Tg mice in which ephrin-B2 was overexpressed in only vascular endothelial cells (ECs). This finding suggests that non-ECs expressing ephrin-B2 alter the migration of ECs expressing EphB receptors into the intersomitic region where ephrin-B2 expression is normally absent. CAGp-ephrin-B2 Tg mice show sudden death at neonatal stages from aortic dissecting aneurysms due to defective recruitment of vascular smooth muscle cells to the ascending aorta. EphB/ephrin-B2 signaling between endothelial cells and surrounding mesenchymal cells plays an essential role in vasculogenesis, angiogenesis, and vessel maturation.