Poglut2/3 double knockout in mice results in neonatal lethality with reduced levels of fibrillin in lung tissues.

Fibrillin microfibrils play a critical role in the formation of elastic fibers, tissue/organ development, and cardiopulmonary function. These microfibrils not only provide structural support and flexibility to tissues, but they also regulate growth factor signaling through a plethora of microfibril-binding proteins in the extracellular space. Mutations in fibrillins are associated with human diseases affecting cardiovascular, pulmonary, skeletal, and ocular systems. Fibrillins consist of up to 47 epidermal growth factor-like repeats, of which more than half are modified by protein O-glucosyltransferase 2 (POGLUT2) and/or POGLUT3. Loss of these modifications reduces secretion of N-terminal fibrillin constructs overexpressed in vitro. Here, we investigated the role of POGLUT2 and POGLUT3 in vivo using a Poglut2/3 double knockout (DKO) mouse model. Blocking O-glucosylation caused neonatal death with skeletal, pulmonary, and eye defects reminiscent of fibrillin/elastin mutations. Proteomic analyses of DKO dermal fibroblast medium and extracellular matrix provided evidence that fibrillins were more sensitive to loss of O-glucose compared to other POGLUT2/3 substrates. This conclusion was supported by immunofluorescent analyses of late gestation DKO lungs where FBN levels were reduced and microfibrils appeared fragmented in the pulmonary arteries and veins, bronchioles, and developing saccules. Defects in fibrillin microfibrils likely contributed to impaired elastic fiber formation and histological changes observed in DKO lung blood vessels, bronchioles, and saccules. Collectively, these results highlight the importance of POGLUT2/3-mediated O-glucosylation in vivo and open the possibility that O-glucose modifications on fibrillin influence microfibril assembly and or protein interactions in the ECM environment.

In chronic kidney disease altered cardiac metabolism precedes cardiac hypertrophy.

Conduit arterial disease in chronic kidney disease (CKD) is an important cause of cardiac complications. Cardiac function in CKD has not been studied in the absence of arterial disease. In an Alport syndrome model bred not to have conduit arterial disease, mice at 225 days of life (dol) had CKD equivalent to humans with CKD stage 4-5. Parathyroid hormone (PTH) and FGF23 levels were one log order elevated, circulating sclerostin was elevated, and renal activin A was strongly induced. Aortic Ca levels were not increased, and vascular smooth muscle cell (VSMC) transdifferentiation was absent. The CKD mice were not hypertensive, and cardiac hypertrophy was absent. Freshly excised cardiac tissue respirometry (Oroboros) showed that ADP-stimulated O2 flux was diminished from 52 to 22 pmol/mg (P = 0.022). RNA-Seq of cardiac tissue from CKD mice revealed significantly decreased levels of cardiac mitochondrial oxidative phosphorylation genes. To examine the effect of activin A signaling, some Alport mice were treated with a monoclonal Ab to activin A or an isotype-matched IgG beginning at 75 days of life until euthanasia. Treatment with the activin A antibody (Ab) did not affect cardiac oxidative phosphorylation. However, the activin A antibody was active in the skeleton, disrupting the effect of CKD to stimulate osteoclast number, eroded surfaces, and the stimulation of osteoclast-driven remodeling. The data reported here show that cardiac mitochondrial respiration is impaired in CKD in the absence of conduit arterial disease. This is the first report of the direct effect of CKD on cardiac respiration.NEW & NOTEWORTHY Heart disease is an important morbidity of chronic kidney disease (CKD). Hypertension, vascular stiffness, and vascular calcification all contribute to cardiac pathophysiology. However, cardiac function in CKD devoid of vascular disease has not been studied. Here, in an animal model of human CKD without conduit arterial disease, we analyze cardiac respiration and discover that CKD directly impairs cardiac mitochondrial function by decreasing oxidative phosphorylation. Protection of cardiac oxidative phosphorylation may be a therapeutic target in CKD.

Changes in transmural mass transport correlate with ascending thoracic aortic aneurysm diameter in a fibulin-4 E57K knockin mouse model.

Thoracic aortic aneurysm is characterized by dilation of the aortic diameter by greater than 50%, which can lead to dissection or rupture. Common histopathology includes extracellular matrix remodeling that may affect transmural mass transport, defined as the movement of fluids and solutes across the wall. We measured in vitro ascending thoracic aorta mass transport in a mouse model with partial aneurysm phenotype penetration due to a mutation in the extracellular matrix protein fibulin-4 [Fbln4E57K/E57K, referred to as MU-A (aneurysm) or MU-NA (no aneurysm)]. To push the aneurysm phenotype, we also included MU mice with reduced levels of lysyl oxidase [Fbln4E57K/E57K;Lox+/-, referred to as MU-XA (extreme aneurysm)] and compared all groups to wild-type (WT) littermates. The phenotype variation allows investigation of how aneurysm severity correlates with mass transport parameters and extracellular matrix organization. We found that MU-NA ascending thoracic aortae have similar hydraulic conductance (Lp) to WT, but 397% higher solute permeability (ω) for 4 kDa FITC-dextran. In contrast, MU-A and MU-XA ascending thoracic aortae have 44-68% lower Lp and similar ω to WT. The results suggest that ascending thoracic aortic aneurysm progression involves an initial increase in ω, followed by a decrease in Lp after the aneurysm has formed. All MU ascending thoracic aortae are longer and have increased elastic fiber fragmentation in the extracellular matrix. There is a negative correlation between diameter and Lp or ω in MU ascending thoracic aortae. Changes in mass transport due to elastic fiber fragmentation could contribute to aneurysm progression or be leveraged for treatment.NEW & NOTEWORTHY Transmural mass transport is quantified in the ascending thoracic aorta of mice with a mutation in fibulin-4 that is associated with thoracic aortic aneurysms. Fluid and solute transport depend on aneurysm severity, correlate with elastic fiber fragmentation, and may be affected by proteoglycan deposition. Transport properties of the ascending thoracic aorta are provided and can be used in computational models. The changes in mass transport may contribute to aneurysm progression or be leveraged for aneurysm treatment.

Endothelial ether lipids link the vasculature to blood pressure, behavior, and neurodegeneration.

Vascular disease contributes to neurodegeneration, which is associated with decreased blood pressure in older humans. Plasmalogens, ether phospholipids produced by peroxisomes, are decreased in Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders. However, the mechanistic links between ether phospholipids, blood pressure, and neurodegeneration are not fully understood. Here, we show that endothelium-derived ether phospholipids affect blood pressure, behavior, and neurodegeneration in mice. In young adult mice, inducible endothelial-specific disruption of PexRAP, a peroxisomal enzyme required for ether lipid synthesis, unexpectedly decreased circulating plasmalogens. PexRAP endothelial knockout (PEKO) mice responded normally to hindlimb ischemia but had lower blood pressure and increased plasma renin activity. In PEKO as compared with control mice, tyrosine hydroxylase was decreased in the locus coeruleus, which maintains blood pressure and arousal. PEKO mice moved less, slept more, and had impaired attention to and recall of environmental events as well as mild spatial memory deficits. In PEKO hippocampus, gliosis was increased, and a plasmalogen associated with memory was decreased. Despite lower blood pressure, PEKO mice had generally normal homotopic functional connectivity by optical neuroimaging of the cerebral cortex. Decreased glycogen synthase kinase-3 phosphorylation, a marker of neurodegeneration, was detected in PEKO cerebral cortex. In a co-culture system, PexRAP knockdown in brain endothelial cells decreased glycogen synthase kinase-3 phosphorylation in co-cultured astrocytes that was rescued by incubation with the ether lipid alkylglycerol. Taken together, our findings suggest that endothelium-derived ether lipids mediate several biological processes and may also confer neuroprotection in mice.

Macrophage angiotensin II type 2 receptor triggers neuropathic pain.

Peripheral nerve damage initiates a complex series of structural and cellular processes that culminate in chronic neuropathic pain. The recent success of a type 2 angiotensin II (Ang II) receptor (AT2R) antagonist in a phase II clinical trial for the treatment of postherpetic neuralgia suggests angiotensin signaling is involved in neuropathic pain. However, transcriptome analysis indicates a lack of AT2R gene (Agtr2) expression in human and rodent sensory ganglia, raising questions regarding the tissue/cell target underlying the analgesic effect of AT2R antagonism. We show that selective antagonism of AT2R attenuates neuropathic but not inflammatory mechanical and cold pain hypersensitivity behaviors in mice. Agtr2-expressing macrophages (MΦs) constitute the predominant immune cells that infiltrate the site of nerve injury. Interestingly, neuropathic mechanical and cold pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral MΦs and AT2R-null hematopoietic cell transplantation. Our study identifies AT2R on peripheral MΦs as a critical trigger for pain sensitization at the site of nerve injury, and therefore proposes a translatable peripheral mechanism underlying chronic neuropathic pain.

Vascular elastic fiber heterogeneity in health and disease.

PURPOSE OF REVIEW: Elastin has historically been described as an amorphous protein that functions to provide recoil to tissues that stretch. However, evidence is growing that elastin's role may not be limited to biomechanics. In this minireview, we will summarize current knowledge regarding vascular elastic fibers, focusing on structural differences along the arterial tree and how those differences may influence the behavior of affiliated cells. RECENT FINDINGS: Regional heterogeneity, including differences in elastic lamellar number, density and cell developmental origin, plays an important role in vessel health and function. These differences impact cell-cell communication, proliferation and movement. Perturbations of normal cell-matrix interactions are correlated with human diseases including aneurysm, atherosclerosis and hypertension. SUMMARY: Although classically described as a structural protein, recent data suggest that differences in elastin deposition along the arterial tree have important effects on heterotypic cell interactions and human disease.

Sodium-activated potassium channels moderate excitability in vascular smooth muscle.

KEY POINTS: We report that a sodium-activated potassium current, IKNa , has been inadvertently overlooked in both conduit and resistance arterial smooth muscle cells. IKNa is a major K+ resting conductance and is absent in cells of IKNa knockout (KO) mice. The phenotype of the IKNa KO is mild hypertension, although KO mice react more strongly than wild-type with raised blood pressure when challenged with vasoconstrictive agents. IKNa is negatively regulated by angiotensin II acting through Gαq protein-coupled receptors. In current clamp, KO arterial smooth muscle cells have easily evoked Ca2+ -dependent action potentials. ABSTRACT: Although several potassium currents have been reported to play a role in arterial smooth muscle (ASM), we find that one of the largest contributors to membrane conductance in both conduit and resistance ASMs has been inadvertently overlooked. In the present study, we show that IKNa , a sodium-activated potassium current, contributes a major portion of macroscopic outward current in a critical physiological voltage range that determines intrinsic cell excitability; IKNa is the largest contributor to ASM cell resting conductance. A genetic knockout (KO) mouse strain lacking KNa channels (KCNT1 and KCNT2) shows only a modest hypertensive phenotype. However, acute administration of vasoconstrictive agents such as angiotensin II (Ang II) and phenylephrine results in an abnormally large increase in blood pressure in the KO animals. In wild-type animals Ang II acting through Gαq protein-coupled receptors down-regulates IKNa , which increases the excitability of the ASMs. The complete genetic removal of IKNa in KO mice makes the mutant animal more vulnerable to vasoconstrictive agents, thus producing a paroxysmal-hypertensive phenotype. This may result from the lowering of cell resting K+ conductance allowing the cells to depolarize more readily to a variety of excitable stimuli. Thus, the sodium-activated potassium current may serve to moderate blood pressure in instances of heightened stress. IKNa may represent a new therapeutic target for hypertension and stroke.

Loss of function mutation in LOX causes thoracic aortic aneurysm and dissection in humans.

Thoracic aortic aneurysms and dissections (TAAD) represent a substantial cause of morbidity and mortality worldwide. Many individuals presenting with an inherited form of TAAD do not have causal mutations in the set of genes known to underlie disease. Using whole-genome sequencing in two first cousins with TAAD, we identified a missense mutation in the lysyl oxidase (LOX) gene (c.893T > G encoding p.Met298Arg) that cosegregated with disease in the family. Using clustered regularly interspaced short palindromic repeats (CRISPR)/clustered regularly interspaced short palindromic repeats-associated protein-9 nuclease (Cas9) genome engineering tools, we introduced the human mutation into the homologous position in the mouse genome, creating mice that were heterozygous and homozygous for the human allele. Mutant mice that were heterozygous for the human allele displayed disorganized ultrastructural properties of the aortic wall characterized by fragmented elastic lamellae, whereas mice homozygous for the human allele died shortly after parturition from ascending aortic aneurysm and spontaneous hemorrhage. These data suggest that a missense mutation in LOX is associated with aortic disease in humans, likely through insufficient cross-linking of elastin and collagen in the aortic wall. Mutation carriers may be predisposed to vascular diseases because of weakened vessel walls under stress conditions. LOX sequencing for clinical TAAD may identify additional mutation carriers in the future. Additional studies using our mouse model of LOX-associated TAAD have the potential to clarify the mechanism of disease and identify novel therapeutics specific to this genetic cause.

Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous, Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities.

Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.

Chronic antihypertensive treatment improves pulse pressure but not large artery mechanics in a mouse model of congenital vascular stiffness.

Increased arterial stiffness is a common characteristic of humans with Williams-Beuren syndrome and mouse models of elastin insufficiency. Arterial stiffness is associated with multiple negative cardiovascular outcomes, including myocardial infarction, stroke, and sudden death. Therefore, identifying therapeutic interventions that improve arterial stiffness in response to changes in elastin levels is of vital importance. The goal of this study was to determine the effect of chronic pharmacologic therapy with different classes of antihypertensive medications on arterial stiffness in elastin insufficiency. Elastin-insufficient mice 4-6 wk of age and wild-type littermates were subcutaneously implanted with osmotic micropumps delivering a continuous dose of one of the following: vehicle, losartan, nicardipine, or propranolol for 8 wk. At the end of treatment period, arterial blood pressure and large artery compliance and remodeling were assessed. Our results show that losartan and nicardipine treatment lowered blood pressure and pulse pressure in elastin-insufficient mice. Elastin and collagen content of abdominal aortas as well as ascending aorta and carotid artery biomechanics were not affected by any of the drug treatments in either genotype. By reducing pulse pressure and shifting the working pressure range of an artery to a more compliant region of the pressure-diameter curve, antihypertensive medications may mitigate the consequences of arterial stiffness, an effect that is drug class independent. These data emphasize the importance of early recognition and long-term management of hypertension in Williams-Beuren syndrome and elastin insufficiency.

Endothelial cell Piezo1 promotes vascular smooth muscle cell differentiation on large arteries.

Vascular stabilization is a mechanosensitive process, in part driven by blood flow. Here, we demonstrate the involvement of the mechanosensitive ion channel, Piezo1, in promoting arterial accumulation of vascular smooth muscle cells (vSMCs) during zebrafish development. Using a series of small molecule antagonists or agonists to temporally regulate Piezo1 activity, we identified a role for the Piezo1 channel in regulating klf2a levels and altered targeting of vSMCs between arteries and veins. Increasing Piezo1 activity suppressed klf2a and increased vSMC association with the cardinal vein, while inhibition of Piezo1 activity increased klf2a levels and decreased vSMC association with arteries. We supported the small molecule data with in vivo genetic suppression of piezo1 and 2 in zebrafish, resulting in loss of transgelin+ vSMCs on the dorsal aorta. Further, endothelial cell (EC)-specific Piezo1 knockout in mice was sufficient to decrease vSMC accumulation along the descending dorsal aorta during development, thus phenocopying our zebrafish data, and supporting functional conservation of Piezo1 in mammals. To determine mechanism, we used in vitro modeling assays to demonstrate that differential sensing of pulsatile versus laminar flow forces across endothelial cells changes the expression of mural cell differentiation genes. Together, our findings suggest a crucial role for EC Piezo1 in sensing force within large arteries to mediate mural cell differentiation and stabilization of the arterial vasculature.

Interference with PPAR gamma function in smooth muscle causes vascular dysfunction and hypertension.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor that plays a critical role in metabolism. Thiazolidinediones, high-affinity PPARgamma ligands used clinically to treat type II diabetes, have been reported to lower blood pressure and provide other cardiovascular benefits. Some mutations in PPARgamma (PPARG) cause type II diabetes and severe hypertension. Here we tested the hypothesis that PPARgamma in vascular muscle plays a role in the regulation of vascular tone and blood pressure. Transgenic mice expressing dominant-negative mutations in PPARgamma under the control of a smooth-muscle-specific promoter exhibit a loss of responsiveness to nitric oxide and striking alterations in contractility in the aorta, hypertrophy and inward remodeling in the cerebral microcirculation, and systolic hypertension. These results identify PPARgamma as pivotal in vascular muscle as a regulator of vascular structure, vascular function, and blood pressure, potentially explaining some of the cardioprotective effects of thiazolidinediones.

A new mouse model of elastin haploinsufficiency highlights the importance of elastin to vascular development and blood pressure regulation.

Supravalvular aortic stenosis (SVAS) is an autosomal dominant disease resulting from elastin (ELN) haploinsufficiency. Individuals with SVAS typically develop a thickened arterial media with an increased number of elastic lamellae and smooth muscle cell (SMC) layers and stenosis superior to the aortic valve. A mouse model of SVAS (Eln+/-) was generated that recapitulates many aspects of the human disease, including increased medial SMC layers and elastic lamellae, large artery stiffness, and hypertension. The vascular changes in these mice were thought to be responsible for the hypertension phenotype. However, a renin gene (Ren) duplication in the original 129/Sv genetic background and carried through numerous strain backcrosses raised the possibility of renin-mediated effects on blood pressure. To exclude excess renin activity as a disease modifier, we utilized the Cre-LoxP system to rederive Eln hemizygous mice on a pure C57BL/6 background (Sox2-Cre;Elnf/f). Here we show that Sox2-Cre;Eln+/f mice, with a single Ren1 gene and normal renin levels, phenocopy the original global knockout line. Characteristic traits include an increased number of elastic lamellae and SMC layers, stiff elastic arteries, and systolic hypertension with widened pulse pressure. Importantly, small resistance arteries of Sox2-Cre;Eln+/f mice exhibit a significant change in endothelial cell function and hypercontractility to angiotensin II, findings that point to pathway-specific alterations in resistance arteries that contribute to the hypertensive phenotype. These data confirm that the cardiovascular changes, particularly systolic hypertension, seen in Eln+/- mice are due to Eln hemizygosity rather than Ren duplication.

Electrophysiology of human iPSC-derived vascular smooth muscle cells and cell autonomous consequences of Cantu Syndrome mutations.

Objective: Cantu Syndrome (CS), a multisystem disease with a complex cardiovascular phenotype, is caused by GoF variants in the Kir6.1/SUR2 subunits of ATP-sensitive potassium (K ATP ) channels, and is characterized by low systemic vascular resistance, as well as tortuous, dilated vessels, and decreased pulse-wave velocity. Thus, CS vascular dysfunction is multifactorial, with distinct hypomyotonic and hyperelastic components. To dissect whether such complexities arise cell-autonomously within vascular smooth muscle cells (VSMCs), or as secondary responses to the pathophysiological milieu, we assessed electrical properties and gene expression in human induced pluripotent stem cell-derived VSMCs (hiPSC-VSMCs), differentiated from control and CS patient-derived hiPSCs, and in native mouse control and CS VSMCs. Approach and Results: Whole-cell voltage-clamp of isolated aortic and mesenteric VSMCs isolated from wild type (WT) and Kir6.1[V65M] (CS) mice revealed no difference in voltage-gated K + (K v ) or Ca 2+ currents. K v and Ca 2+ currents were also not different between validated hiPSC-VSMCs differentiated from control and CS patient-derived hiPSCs. Pinacidil-sensitive K ATP currents in control hiPSC-VSMCs were consistent with those in WT mouse VSMCs, and were considerably larger in CS hiPSC-VSMCs. Consistent with lack of any compensatory modulation of other currents, this resulted in membrane hyperpolarization, explaining the hypomyotonic basis of CS vasculopathy. Increased compliance and dilation in isolated CS mouse aortae, was associated with increased elastin mRNA expression. This was consistent with higher levels of elastin mRNA in CS hiPSC-VSMCs, suggesting that the hyperelastic component of CS vasculopathy is a cell-autonomous consequence of vascular K ATP GoF. Conclusions: The results show that hiPSC-VSMCs reiterate expression of the same major ion currents as primary VSMCs, validating the use of these cells to study vascular disease. The results further indicate that both the hypomyotonic and hyperelastic components of CS vasculopathy are cell-autonomous phenomena driven by K ATP overactivity within VSMCs.

SVEP1 is an endogenous ligand for the orphan receptor PEAR1.

Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 (SVEP1) is an extracellular matrix protein that causally promotes vascular disease and associates with platelet reactivity in humans. Here, using a human genomic and proteomic approach, we identify a high affinity, disease-relevant, and potentially targetable interaction between SVEP1 and the orphan receptor Platelet and Endothelial Aggregation Receptor 1 (PEAR1). This interaction promotes PEAR1 phosphorylation and disease associated AKT/mTOR signaling in vascular cells and platelets. Mice lacking SVEP1 have reduced platelet activation, and exogenous SVEP1 induces PEAR1-dependent activation of platelets. SVEP1 and PEAR1 causally and concordantly relate to platelet phenotypes and cardiovascular disease in humans, as determined by Mendelian Randomization. Targeting this receptor-ligand interaction may be a viable therapeutic strategy to treat or prevent cardiovascular and thrombotic disease.

Brain-selective overexpression of human Angiotensin-converting enzyme type 2 attenuates neurogenic hypertension.

RATIONALE: Angiotensin converting enzyme type 2 (ACE2) is a new member of the brain renin-angiotensin system, that might be activated by an overactive renin-angiotensin system. OBJECTIVE: To clarify the role of central ACE2 using a new transgenic mouse model with human (h)ACE2 under the control of a synapsin promoter, allowing neuron-targeted expression in the central nervous system. METHODS AND RESULTS: Syn-hACE2 (SA) transgenic mice exhibit high hACE2 protein expression and activity throughout the brain. Baseline hemodynamic parameters (telemetry), autonomic function, and spontaneous baroreflex sensitivity (SBRS) were not significantly different between SA mice and nontransgenic littermates. Brain-targeted ACE2 overexpression attenuated the development of neurogenic hypertension (Ang II infusion: 600 ng/kg per minute for 14 days) and the associated reduction of both SBRS and parasympathetic tone. This prevention of hypertension by ACE2 overexpression was reversed by blockade of the Ang-(1-7) receptor (d-Ala7-Ang-[1-7]; 600 ng/kg per minute). Brain angiotensin II type 2 (AT(2))/AT(1) and Mas/AT(1) receptor ratios were significantly increased in SA mice. They remained higher following Ang II infusion but were dramatically reduced after Ang-(1-7) receptor blockade. ACE2 overexpression resulted in increased NOS and NO levels in the brain, and prevented the Ang II-mediated decrease in NOS expression in regions modulating blood pressure regulation. CONCLUSIONS: ACE2 overexpression attenuates the development of neurogenic hypertension partially by preventing the decrease in both SBRS and parasympathetic tone. These protective effects might be mediated by enhanced NO release in the brain resulting from Mas and AT(2) receptor upregulation. Taken together, our data highlight the compensatory role of central ACE2 and its potential benefits as a therapeutic target for neurogenic hypertension.

Labor induction with oxytocin in pregnant rats is not associated with oxidative stress in the fetal brain.

Despite the widespread use of oxytocin for induction of labor, mechanistic insights into fetal/neonatal wellbeing are lacking because of the absence of an animal model that recapitulates modern obstetric practice. Here, we create and validate a hi-fidelity pregnant rat model that mirrors labor induction with oxytocin in laboring women. The model consists of an implantable preprogrammed microprocessor-controlled infusion pump that delivers a gradually escalating dose of intravenous oxytocin to induce birth at term gestation. We validated the model with molecular biological experiments on the uterine myometrium and telemetry-supported assessment of changes in intrauterine pressure. Finally, we applied this model to test the hypothesis that labor induction with oxytocin would be associated with oxidative stress in the newborn brain. Analysis of biomarkers of oxidative stress and changes in the expression of associated genes were no different between oxytocin-exposed and saline-treated pups, suggesting that oxytocin-induced labor was not associated with oxidative stress in the developing brain. Collectively, we provide a viable and realistic animal model for labor induction and augmentation with oxytocin that would enable new lines of investigation related to the impact of perinatal oxytocin exposure on the mother-infant dyad.

The ZFP36 family of RNA binding proteins regulates homeostatic and autoreactive T cell responses.

RNA binding proteins are important regulators of T cell activation, proliferation, and cytokine production. The zinc finger protein 36 (ZFP36) family genes (Zfp36, Zfp36l1, and Zfp36l2) encode RNA binding proteins that promote the degradation of transcripts containing AU-rich elements. Numerous studies have demonstrated both individual and shared functions of the ZFP36 family in immune cells, but their collective function in T cells remains unclear. Here, we found a redundant and critical role for the ZFP36 proteins in regulating T cell quiescence. T cell-specific deletion of all three ZFP36 family members in mice resulted in early lethality, immune cell activation, and multiorgan pathology characterized by inflammation of the eyes, central nervous system, kidneys, and liver. Mice with T cell-specific deletion of any two Zfp36 genes were protected from this spontaneous syndrome. Triply deficient T cells overproduced proinflammatory cytokines, including IFN-γ, TNF, and GM-CSF, due to increased mRNA stability of these transcripts. Unexpectedly, T cell-specific deletion of both Zfp36l1 and Zfp36l2 rendered mice resistant to experimental autoimmune encephalomyelitits due to failed priming of antigen-specific CD4+ T cells. ZFP36L1 and ZFP36L2 double-deficient CD4+ T cells had poor proliferation during in vitro T helper cell polarization. Thus, the ZFP36 family redundantly regulates T cell quiescence at homeostasis, but ZFP36L1 and ZFP36L2 are specifically required for antigen-specific T cell clonal expansion.

Bioinformatic analysis of gene sets regulated by ligand-activated and dominant-negative peroxisome proliferator-activated receptor gamma in mouse aorta.

OBJECTIVE: Drugs that activate peroxisome proliferator-activated receptor (PPAR) gamma improve glucose sensitivity and lower blood pressure, whereas dominant-negative mutations in PPARgamma cause severe insulin resistance and hypertension. We hypothesize that these PPARgamma mutants regulate target genes opposite to those of ligand-mediated activation, and we tested this hypothesis on a genomewide scale. METHODS AND RESULTS: We integrated gene expression data in aorta specimens from mice treated with the PPARgamma ligand rosiglitazone with data from mice containing a globally expressed knockin of the PPARgamma P465L dominant-negative mutation. We also integrated our data with publicly available data sets containing the following: (1) gene expression profiles in many human tissues, (2) PPARgamma target genes in 3T3-L1 adipocytes, and (3) experimentally validated PPARgamma binding sites throughout the genome. Many classic PPARgamma target genes were induced by rosiglitazone and repressed by dominant-negative PPARgamma. A similar pattern was observed for about 90% of the gene sets regulated by both rosiglitazone and dominant-negative PPARgamma. Genes exhibiting this pattern of contrasting regulation were significantly enriched for nearby PPARgamma binding sites. CONCLUSIONS: These results provide convincing evidence that the PPARgamma P465L mutation causes transcriptional effects that are opposite to those mediated by PPARgamma ligand, thus validating mice carrying the mutation as a model of PPARgamma interference.