Complementary Role of Oxytocin and Vasopressin in Cardiovascular Regulation.

The neurons secreting oxytocin (OXY) and vasopressin (AVP) are located mainly in the supraoptic, paraventricular, and suprachiasmatic nucleus of the brain. Oxytocinergic and vasopressinergic projections reach several regions of the brain and the spinal cord. Both peptides are released from axons, soma, and dendrites and modulate the excitability of other neuroregulatory pathways. The synthesis and action of OXY and AVP in the peripheral organs (eye, heart, gastrointestinal system) is being investigated. The secretion of OXY and AVP is influenced by changes in body fluid osmolality, blood volume, blood pressure, hypoxia, and stress. Vasopressin interacts with three subtypes of receptors: V1aR, V1bR, and V2R whereas oxytocin activates its own OXTR and V1aR receptors. AVP and OXY receptors are present in several regions of the brain (cortex, hypothalamus, pons, medulla, and cerebellum) and in the peripheral organs (heart, lungs, carotid bodies, kidneys, adrenal glands, pancreas, gastrointestinal tract, ovaries, uterus, thymus). Hypertension, myocardial infarction, and coexisting factors, such as pain and stress, have a significant impact on the secretion of oxytocin and vasopressin and on the expression of their receptors. The inappropriate regulation of oxytocin and vasopressin secretion during ischemia, hypoxia/hypercapnia, inflammation, pain, and stress may play a significant role in the pathogenesis of cardiovascular diseases.

Peripheral Vascular Abnormalities in Anorexia Nervosa: A Psycho-Neuro-Immune-Metabolic Connection.

Immune, neuroendocrine, and autonomic nervous system dysregulation in anorexia nervosa lead to cardiovascular complications that can potentially result in increased morbidity and mortality. It is suggested that a complex non-invasive assessment of cardiovascular autonomic regulation-cardiac vagal control, sympathetic vascular activity, and cardiovascular reflex control-could represent a promising tool for early diagnosis, personalized therapy, and monitoring of therapeutic interventions in anorexia nervosa particularly at a vulnerable adolescent age. In this view, we recommend to consider in the diagnostic route, at least in the subset of patients with peripheral microvascular symptoms, a nailfold video-capillaroscopy as an easy not invasive tool for the early assessing of possible cardiovascular involvement.

Reduced dosage of ß-catenin provides significant rescue of cardiac outflow tract anomalies in a Tbx1 conditional null mouse model of 22q11.2 deletion syndrome.

The 22q11.2 deletion syndrome (22q11.2DS; velo-cardio-facial syndrome; DiGeorge syndrome) is a congenital anomaly disorder in which haploinsufficiency of TBX1, encoding a T-box transcription factor, is the major candidate for cardiac outflow tract (OFT) malformations. Inactivation of Tbx1 in the anterior heart field (AHF) mesoderm in the mouse results in premature expression of pro-differentiation genes and a persistent truncus arteriosus (PTA) in which septation does not form between the aorta and pulmonary trunk. Canonical Wnt/ß-catenin has major roles in cardiac OFT development that may act upstream of Tbx1. Consistent with an antagonistic relationship, we found the opposite gene expression changes occurred in the AHF in ß-catenin loss of function embryos compared to Tbx1 loss of function embryos, providing an opportunity to test for genetic rescue. When both alleles of Tbx1 and one allele of ß-catenin were inactivated in the Mef2c-AHF-Cre domain, 61% of them (n = 34) showed partial or complete rescue of the PTA defect. Upregulated genes that were oppositely changed in expression in individual mutant embryos were normalized in significantly rescued embryos. Further, ß-catenin was increased in expression when Tbx1 was inactivated, suggesting that there may be a negative feedback loop between canonical Wnt and Tbx1 in the AHF to allow the formation of the OFT. We suggest that alteration of this balance may contribute to variable expressivity in 22q11.2DS.

Integrin ß1 activity is required for cardiovascular formation in zebrafish.

Integrins are transmembrane molecules that facilitate cell-to-cell and cell-to-extracellular matrix (ECM) interactions. Integrin molecules are heterodimers that consist of α- and ß-subunits. The integrin ß1 gene is widely expressed in vivo and is the major ß molecule in many tissues; however, tissue-specific roles of integrin ß1 are still elusive. In this study, we investigated integrin ß1 function in endothelial cells of zebrafish. An integrin ß1b mutant zebrafish exhibited morphological abnormalities in blood vessel formation, cephalic hemorrhage and a decreased responsiveness to tactile stimulation during development. To determine the role of integrin ß1b in vascular formation, we developed a Gal4/UAS-mediated conditional inactivation of integrin ß1 by expressing the cytoplasmic region of integrin ß1 that acts as a dominant-negative (DN) isoform. Expression of integrin ß1 DN in endothelial cells induced blood vessel abnormalities as in integrin ß1b mutants. These results show that endothelial cells require integrin activity for the formation and/or maintenance of blood vessels in zebrafish. Furthermore, our time-lapse recording visualized the breakpoint of cephalic vessels and the hemorrhage onset. Taken together, our tissue-specific inactivation of integrin ß1 in zebrafish is powerful tools for functional analysis of integrin ß1 in developing tissues.

Constitutive activation of the PI3K-AKT pathway and cardiovascular abnormalities in an individual with Kosaki overgrowth syndrome.

Kosaki overgrowth syndrome is a recently described syndrome characterized by distinctive facial features, brain white matter lesions, and developmental delay. Germline activating heterozygous PDGFRB mutations have been reported in this condition. Systemic connective tissue-type findings have been described in some individuals. We describe a 19-year-old Caucasian female with a history of hydrocephalus, Dandy-Walker malformation, cervical spine arachnoid cyst, progressive scoliosis, and overgrowth. Her physical exam included distinctive craniofacial dysmorphism, as well as soft and hyperextensible skin. Cardiovascular imaging during adolescence revealed saccular aneurysms in both coronary artery systems and subtle tortuosity of the cervical vertebral arteries. Exome sequencing trio analysis identified a de novo previously reported pathogenic variant in PDGFRB, c.1696T>C (p.[Trp566Arg]). Further functional studies included platelet-derived growth factor cellular metabolic pathway activity that confirmed the variant causes a constitutive activation of the PI3K-AKT pathway. This is the first report to characterize the activating nature of this PDGFRB variant. We also highlight the connective tissue findings seen in Kosaki overgrowth syndrome and recommend baseline echocardiographic evaluation in all individuals with this condition with particular emphasis on coronary arteries.

GLUT10 is required for the development of the cardiovascular system and the notochord and connects mitochondrial function to TGFß signaling.

Growth factor signaling results in dramatic phenotypic changes in cells, which require commensurate alterations in cellular metabolism. Mutations in SLC2A10/GLUT10, a member of the facilitative glucose transporter family, are associated with altered transforming growth factor-ß (TGFß) signaling in patients with arterial tortuosity syndrome (ATS). The objective of this work was to test whether SLC2A10/GLUT10 can serve as a link between TGFß-related transcriptional regulation and metabolism during development. In zebrafish embryos, knockdown of slc2a10 using antisense morpholino oligonucleotide injection caused a wavy notochord and cardiovascular abnormalities with a reduced heart rate and blood flow, which was coupled with an incomplete and irregular vascular patterning. This was phenocopied by treatment with a small-molecule inhibitor of TGFß receptor (tgfbr1/alk5). Array hybridization showed that the changes at the transcriptome level caused by the two treatments were highly correlated, revealing that a reduced tgfbr1 signaling is a key feature of ATS in early zebrafish development. Interestingly, a large proportion of the genes, which were specifically dysregulated after glut10 depletion gene and not by tgfbr1 inhibition, play a major role in mitochondrial function. Consistent with these results, slc2a10 morphants showed decreased respiration and reduced TGFß reporter gene activity. Finally, co-injection of antisense morpholinos targeting slc2a10 and smad7 (a TGFß inhibitor) resulted in a partial rescue of smad7 morphant phenotypes, suggesting scl2a10/glut10 functions downstream of smads. Taken together, glut10 is essential for cardiovascular development by facilitating both mitochondrial respiration and TGFß signaling.

Regulation of mammalian Notch signaling and embryonic development by the protein O-glucosyltransferase Rumi.

Protein O-glucosylation is a conserved post-translational modification that occurs on epidermal growth factor-like (EGF) repeats harboring the C(1)-X-S-X-P-C(2) consensus sequence. The Drosophila protein O-glucosyltransferase (Poglut) Rumi regulates Notch signaling, but the contribution of protein O-glucosylation to mammalian Notch signaling and embryonic development is not known. Here, we show that mouse Rumi encodes a Poglut, and that Rumi(-/-) mouse embryos die before embryonic day 9.5 with posterior axis truncation and severe defects in neural tube development, somitogenesis, cardiogenesis and vascular remodeling. Rumi knockdown in mouse cell lines results in cellular and molecular phenotypes of loss of Notch signaling without affecting Notch ligand binding. Biochemical, cell culture and cross-species transgenic experiments indicate that a decrease in Rumi levels results in reduced O-glucosylation of Notch EGF repeats, and that the enzymatic activity of Rumi is key to its regulatory role in the Notch pathway. Genetic interaction studies show that removing one copy of Rumi in a Jag1(+/-) (jagged 1) background results in severe bile duct morphogenesis defects. Altogether, our data indicate that addition of O-glucose to EGF repeats is essential for mouse embryonic development and Notch signaling, and that Jag1-induced signaling is sensitive to the gene dosage of the protein O-glucosyltransferase Rumi. Given that Rumi(-/-) embryos show more severe phenotypes compared to those displayed by other global regulators of canonical Notch signaling, Rumi is likely to have additional important targets during mammalian development.

RBP4 disrupts vitamin A uptake homeostasis in a STRA6-deficient animal model for Matthew-Wood syndrome.

The cellular uptake of vitamin A from its RBP4-bound circulating form (holo-RBP4) is a homeostatic process that evidently depends on the multidomain membrane protein STRA6. In humans, mutations in STRA6 are associated with Matthew-Wood syndrome, manifested by multisystem developmental malformations. Here we addressed the metabolic basis of this inherited disease. STRA6-dependent transfer of retinol from RBP4 into cultured NIH 3T3 fibroblasts was enhanced by lecithin:retinol acyltransferase (LRAT). The retinol transfer was bidirectional, strongly suggesting that STRA6 acts as a retinol channel/transporter. Loss-of-function analysis in zebrafish embryos revealed that Stra6 deficiency caused vitamin A deprivation of the developing eyes. We provide evidence that, in the absence of Stra6, holo-Rbp4 provokes nonspecific vitamin A excess in several embryonic tissues, impairing retinoic acid receptor signaling and gene regulation. These fatal consequences of Stra6 deficiency, including craniofacial and cardiac defects and microphthalmia, were largely alleviated by reducing embryonic Rbp4 levels by morpholino oligonucleotide or pharmacological treatments.

Activin-A in diabetes-induced cardiac malformations in embryos.

BACKGROUND: Heart defects are the most common abnormalities in infants of diabetic mothers. Cardiac malformation is associated with altered expression of the genes in the transforming growth factor ß system, including inhibin ßA, which forms activin-A as a homodimer and functions through its effectors, Smad2 and Smad3. This study aimed to investigate the role of activin-A in diabetes-induced cardiac malformations. METHODS: Diabetes mellitus in female mice (C57BL/6J) was induced via intravenous injection of streptozotocin. The expression of inhibin ßA protein and phosphorylation of Smad2 and Smad3 in the embryonic hearts were examined using immunohistochemical, in situ proximity ligation, and immunoblot assays. Embryos and endocardial cushions of nondiabetic mice were cultured in a high concentration of glucose and treated with activin-A. Mitosis was examined using BrdU incorporation assay and immunohistochemistry of phosphorylated histone H3. Migration of the endocardial cells was assessed using a collagen-based cell migration assay. RESULTS: The levels of inhibin ßA expression and Smad2 and Smad3 activation were significantly reduced by maternal diabetes. Treatment with activin-A significantly increased cell proliferation in the myocardium and migration of endocardial cells, compared with those in vehicle-treated high glucose group, to the level in the euglycemic control group. CONCLUSIONS: Maternal diabetes suppresses the expression of inhibin ßA protein, as well as the activation of Smad2 and Smad3. Activin-A rescues cell proliferation in the myocardium and migration of the endocardial cells suppressed by hyperglycemia. The activin-Smad2/3 signaling system appears to play a role in cardiac malformation in diabetic embryopathy.

Kaempferol protects against cardiovascular abnormalities induced by nitric oxide deficiency in rats by suppressing the TNF-α pathway.

Kaempferol is a natural flavonoid compound that exhibits various pharmacological actions. However, there are few reports regarding the role of kaempferol in cardiovascular abnormalities. This study aimed to assess whether kaempferol could prevent cardiovascular malfunction and hypertrophy provoked by chronic inhibition of nitric oxide (NO) formation in rats. Rats (180-200 g) were treated daily with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) (40 mg/kg, in drinking water) for five weeks concomitant with kaempferol (oral administration) at a dose of 20 mg/kg or 40 mg/kg or lisinopril (5 mg/kg). Kaempferol partially prevented the progression of hypertension provoked by NO inhibition (p < 0.05). Left ventricular malfunction and hypertrophy present in hypertensive rats were alleviated by concurrent administration of kaempferol (p < 0.05). Furthermore, L-NAME rats had increased sympathetic nerve-mediated vasoconstriction and decreased acetylcholine-induced vasorelaxation and aortic wall thickening, which were resolved by kaempferol treatment (p < 0.05). Kaempferol restored tissue superoxide formation, malondialdehyde, catalase activity, plasma nitric oxide metabolites, tumor necrosis factor-alpha (TNF-α) and interleukin-6 in L-NAME rats (p < 0.05). Overexpression of tumor necrosis factor receptor 2 (TNFR2), phosphatidylinositol 3-kinases (PI3K), AKT serine/threonine kinase 1 (Akt1) and smad2/3 in heart tissue and upregulation of tumor necrosis factor receptor 1 (TNFR1), phosphorylated nuclear factor-kappaB (p-NF-κB) and transforming growth factor beta 1 (TGF-ß1) in vascular tissue were suppressed by kaempferol (p < 0.05). In conclusion, kaempferol exerts antihypertensive, cardioprotective, antioxidant, and anti-inflammatory effects in NO-dependent hypertensive rats. The underlying mechanisms of kaempferol in preventing cardiovascular changes induced by L-NAME were due to the suppression of the TNF-α pathway.

Polymorphisms in folate-metabolizing genes and risk of having an offspring with congenital anomalies in the West Siberian region of Russia: a case-control study.

OBJECTIVE: Periconceptional folate supplementation prevents a number of congenital anomalies (CA). The aim of our study was to investigate the association of 11 polymorphisms in the folate-metabolizing genes with the risk of having an offspring with CA in the Russian ethnic group. METHOD: We genotyped 280 mothers having a CA-affected pregnancy and 390 control mothers. The most common malformations among the cases were CA of the nervous, urinary, and cardiovascular systems, and these groups were analyzed separately. RESULTS: In the whole group of CA, we revealed the associations of MTHFR C677T and MTR A2756G loci with increased risk of CA-affected pregnancy. In the group of CA of the cardiovascular system, we observed an association of MTHFR A1298C with decreased risk and an association of MTR A2756G with increased risk of CA. After the Bonferroni correction, only the association between the genotype MTR 2756GG and the risk of having a fetus with CA of the cardiovascular system remained statistically significant (OR = 4.99, P = 0.03). CONCLUSION: These findings indicate that locus A2756G in the MTR gene may play a role in susceptibility to CA of the cardiovascular system in West Siberia, but further research is necessary to confirm the association.

Distinct function of 2 chromatin remodeling complexes that share a common subunit, Williams syndrome transcription factor (WSTF).

A number of nuclear complexes modify chromatin structure and operate as functional units. However, the in vivo role of each component within the complexes is not known. ATP-dependent chromatin remodeling complexes form several types of protein complexes, which reorganize chromatin structure cooperatively with histone modifiers. Williams syndrome transcription factor (WSTF) was biochemically identified as a major subunit, along with 2 distinct complexes: WINAC, a SWI/SNF-type complex, and WICH, an ISWI-type complex. Here, WSTF(-/-) mice were generated to investigate its function in chromatin remodeling in vivo. Loss of WSTF expression resulted in neonatal lethality, and all WSTF(-/-) neonates and approximately 10% of WSTF(+/-) neonates suffered cardiovascular abnormalities resembling those found in autosomal-dominant Williams syndrome patients. Developmental analysis of WSTF(-/-) embryos revealed that Gja5 gene regulation is aberrant from E9.5, conceivably because of inappropriate chromatin reorganization around the promoter regions where essential cardiac transcription factors are recruited. In vitro analysis in WSTF(-/-) mouse embryonic fibroblast (MEF) cells also showed impaired transactivation functions of cardiac transcription activators on the Gja5 promoter, but the effects were reversed by overexpression of WINAC components. Likewise in WSTF(-/-) MEF cells, recruitment of Snf2h, an ISWI ATPase, to PCNA and cell survival after DNA damage were both defective, but were ameliorated by overexpression of WICH components. Thus, the present study provides evidence that WSTF is shared and is a functionally indispensable subunit of the WICH complex for DNA repair and the WINAC complex for transcriptional control.

ß8 integrin and band 4.1B cooperatively regulate morphogenesis of the embryonic heart.

Morphogenesis of the heart is regulated by various cues, including growth factors and extracellular matrix (ECM) proteins. The mechanisms by which cardiac cells properly integrate these cues to regulate growth, differentiation, and migration remain poorly understood. Here we have used genetic strategies in mice to identify αvß8 integrin and its cytoskeletal adaptor protein, Band 4.1B, as essential regulators of cardiac morphogenesis. We demonstrate that approximately 60% of mouse embryos genetically null for ß8 integrin and Band 4.1B display cardiovascular phenotypes and die by E11.5. This premature death is due, in part, to defective development of the cardiac outflow tract (OFT), with reduced expression of smooth muscle α-actin (SMAα-actin) in OFT cells derived from the cardiac neural crest. These data are the first to identify cell adhesion and signaling pathways regulated by αvß8 integrin and Band 4.1B as essential for normal formation and function of the heart during embryogenesis.

Identification and functional characterization of NODAL rare variants in heterotaxy and isolated cardiovascular malformations.

NODAL and its signaling pathway are known to play a key role in specification and patterning of vertebrate embryos. Mutations in several genes encoding components of the NODAL signaling pathway have previously been implicated in the pathogenesis of human left-right (LR) patterning defects. Therefore, NODAL, a member of TGF-beta superfamily of developmental regulators, is a strong candidate to be functionally involved in congenital LR axis patterning defects or heterotaxy. Here we have investigated whether variants in NODAL are present in patients with heterotaxy and/or isolated cardiovascular malformations (CVM) thought to be caused by abnormal heart tube looping. Analysis of a large cohort of cases (n = 269) affected with either classic heterotaxy or looping CVM revealed four different missense variants, one in-frame insertion/deletion and two conserved splice site variants in 14 unrelated subjects (14/269, 5.2%). Although similar with regard to other associated defects, individuals with the NODAL mutations had a significantly higher occurrence of pulmonary valve atresia (P = 0.001) compared with cases without a detectable NODAL mutation. Functional analyses demonstrate that the missense variant forms of NODAL exhibit significant impairment of signaling as measured by decreased Cripto (TDGF-1) co-receptor-mediated activation of artificial reporters. Expression of these NODAL proteins also led to reduced induction of Smad2 phosphorylation and impaired Smad2 nuclear import. Taken together, these results support a role for mutations and rare deleterious variants in NODAL as a cause for sporadic human LR patterning defects.

Induced chromosome deletion in a Williams-Beuren syndrome mouse model causes cardiovascular abnormalities.

AIMS: The Williams-Beuren syndrome (WBS) is a genetic disorder caused by a heterozygous ~1.5-Mb deletion. The aim of this study was to determine how the genetic changes in a Wbs mouse model alter Eln expression, blood pressure, vessel structure, and abdominal aortic wall dynamics in vivo. METHODS: Elastin (ELN) transcript levels were quantified by qRT-PCR and blood pressure was measured with a tail cuff system. M-mode ultrasound was used to track pulsatile abdominal aortic wall motion. Aortas were sectioned and stained to determine medial lamellar structure. RESULTS: ELN transcript levels were reduced by 38-41% in Wbs mice lacking one copy of the ELN gene. These mice also had a 10-20% increase in mean blood pressure and significantly reduced circumferential cyclic strain (p < 0.001). Finally, histological sections showed disorganized and fragmented elastin sheets in Wbs mice, but not the characteristic increase in lamellar units seen in Eln(+/-) mice. CONCLUSIONS: The deletion of Eln in this Wbs mouse model results in lower gene expression, hypertension, reduced cyclic strain, and fragmented elastin sheets. The observation that the number of medial lamellar units is normal in Wbs deletion mice, which is in contrast to Eln(+/-) mice, suggests other genes may be involved in vascular development.

Identification of developmentally toxic drinking water disinfection byproducts and evaluation of data relevant to mode of action.

Reactions between chemicals used to disinfect drinking water and compounds present in source waters produce chemical mixtures containing hundreds of disinfection byproducts (DBPs). Although the results have been somewhat inconsistent, some epidemiological studies suggest associations may exist between DBP exposures and adverse developmental outcomes. The potencies of individual DBPs in rodent and rabbit developmental bioassays suggest that no individual DBP can account for the relative risk estimates reported in the positive epidemiologic studies, leading to the hypothesis that these outcomes could result from the toxicity of DBP mixtures. As a first step in a mixtures risk assessment for DBP developmental effects, this paper identifies developmentally toxic DBPs and examines data relevant to the mode of action (MOA) for DBP developmental toxicity. We identified 24 developmentally toxic DBPs and four adverse developmental outcomes associated with human DBP exposures: spontaneous abortion, cardiovascular defects, neural tube defects, and low birth weight infancy. A plausible MOA, involving hormonal disruption of pregnancy, is delineated for spontaneous abortion, which some epidemiologic studies associate with total trihalomethane and bromodichloromethane exposures. The DBP data for the other three outcomes were inadequate to define key MOA steps.

The N-end rule pathway as a nitric oxide sensor controlling the levels of multiple regulators.

The conjugation of arginine to proteins is a part of the N-end rule pathway of protein degradation. Three amino (N)-terminal residues--aspartate, glutamate and cysteine--are arginylated by ATE1-encoded arginyl-transferases. Here we report that oxidation of N-terminal cysteine is essential for its arginylation. The in vivo oxidation of N-terminal cysteine, before its arginylation, is shown to require nitric oxide. We reconstituted this process in vitro as well. The levels of regulatory proteins bearing N-terminal cysteine, such as RGS4, RGS5 and RGS16, are greatly increased in mouse ATE1-/- embryos, which lack arginylation. Stabilization of these proteins, the first physiological substrates of mammalian N-end rule pathway, may underlie cardiovascular defects in ATE1-/- embryos. Our findings identify the N-end rule pathway as a new nitric oxide sensor that functions through its ability to destroy specific regulatory proteins bearing N-terminal cysteine, at rates controlled by nitric oxide and apparently by oxygen as well.

Gab family proteins are essential for postnatal maintenance of cardiac function via neuregulin-1/ErbB signaling.

Grb2-associated binder (Gab) family of scaffolding adaptor proteins coordinate signaling cascades downstream of growth factor and cytokine receptors. In the heart, among EGF family members, neuregulin-1beta (NRG-1beta, a paracrine factor produced from endothelium) induced remarkable tyrosine phosphorylation of Gab1 and Gab2 via erythroblastic leukemia viral oncogene (ErbB) receptors. We examined the role of Gab family proteins in NRG-1beta/ErbB-mediated signal in the heart by creating cardiomyocyte-specific Gab1/Gab2 double knockout mice (DKO mice). Although DKO mice were viable, they exhibited marked ventricular dilatation and reduced contractility with aging. DKO mice showed high mortality after birth because of heart failure. In addition, we noticed remarkable endocardial fibroelastosis and increase of abnormally dilated vessels in the ventricles of DKO mice. NRG-1beta induced activation of both ERK and AKT in the hearts of control mice but not in those of DKO mice. Using DNA microarray analysis, we found that stimulation with NRG-1beta upregulated expression of an endothelium-stabilizing factor, angiopoietin 1, in the hearts of control mice but not in those of DKO mice, which accounted for the pathological abnormalities in the DKO hearts. Taken together, our observations indicated that in the NRG-1beta/ErbB signaling, Gab1 and Gab2 of the myocardium are essential for both maintenance of myocardial function and stabilization of cardiac capillary and endocardial endothelium in the postnatal heart.

Phenotypic features of ciliary dyskinesia among patients with congenital cardiovascular malformations.

BACKGROUND: Cilia are cell membrane-bound organelles responsible for airway mucus clearance, establishment of left-right organ asymmetry, cardiogenesis, and many other functions in utero. Phenotypic features suggestive of respiratory ciliary dyskinesia among patients with heterotaxy syndrome, defined as complex cardiovascular malformations (CVM) and situs ambiguus (SA), has not been adequately explored. OBJECTIVES: We hypothesized that there is a greater incidence of phenotypic features consistent with ciliary dyskinesia among patients with heterotaxy syndrome compared to patients with other CVM and laterality defects without heterotaxy syndrome. METHODS: Thirty six subjects were identified by medical record search and divided into four groups based on situs status and type of CVM as follows: SA and complex CVM (group 1); SA and simple CVM (group 2); situs solitus and complex CVM (group 3); and situs solitus and simple CVM (group 4). Phenotype was assessed with a clinical questionnaire, nasal nitric oxide (NO) level, and pulmonary function testing. Those with complex CVM underwent additional testing for variants in genes involved in ciliary structure and function. RESULTS: The mean nasal NO level was significantly lower among all subjects with complex CVM regardless of situs anomalies (groups 1 and 3). There was no significant difference in respiratory symptoms or lung function among the four groups. No bi-allelic genetic mutations were detected among patients with complex CVM. CONCLUSIONS: This study identified a relatively lower mean nasal NO level, suggestive of relative ciliary dyskinesia, among subjects with complex CVM. Pulmonary function and clinical symptoms did not reflect significant pulmonary disease among those with complex CVM.

Macrophage Depletion Improves Endothelial Insulin Resistance and Protects against Cardiovascular Injury in Salt-Sensitive Hypertension.

Vascular endothelial insulin signaling is critical for the maintenance of vascular and metabolic homeostasis. We have previously shown that in hypertensive Dahl rats, impaired vascular insulin action is linked to angiotensin II activation of the NFκB inflammatory pathway. Macrophage polarization (M1) has implicated in hypertensive and metabolic diseases. Here, we investigated the effect of macrophage depletion using liposome-encapsulated clodronate (LEC) on endothelial insulin resistance and cardiovascular remodeling in Dahl salt-sensitive (DS) rats. High salt intake (HS) for 5 weeks increased systolic blood pressure (SBP: 192 ± 5 vs. 144 ± 4 mmHg in NS, p < 0.05), aortic and cardiac hypertrophy, cardiac fibrosis, and impaired acetylcholine- and insulin-induced vasorelaxation, accompanied by impaired insulin activation of endothelial nitric oxide synthases (eNOS)/NO signaling. HS rats had a significant increase in CD68 (a monocyte/macrophage marker) expression in the aorta and the heart. LEC reduced SBP (168 ± 5 mmHg, p < 0.05) and cardiovascular injury and improved acetylcholine- and insulin-mediated vasorelaxation and insulin signaling molecules with a reduction in the macrophage infiltration in the aorta and the heart. HS rats also manifested an increase in the aortic expressions of inflammatory cytokines, including the ratio of phosphorylated inhibitory kappa B (Iκb)/Iκb, tumor necrosis factor α, and phosphorylated c-Jun N-terminal kinase (JNK) and oxidative stress, which were reduced in HS/LEC rats. Our results suggest that in salt-sensitive hypertension, macrophage may importantly contribute to endothelial insulin resistance, vascular inflammation, and injury. These findings support the idea that macrophages may be a new target for immunotherapy of vasculopathy in hypertensive and metabolic disorders.