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High-fat diet (HFD)-induced obesity is associated with accumulation of inflammatory cells predominantly in visceral adipose depots [visceral adipose tissue (VAT)] rather than in subcutaneous ones [subcutaneous adipose tissue (SAT)]. The cellular and molecular mechanisms responsible for this phenotypic difference remain poorly understood. Controversy also exists on the overall impact that adipose tissue inflammation has on metabolic health in diet-induced obesity. The endothelium of the microcirculation regulates both the transport of lipids and the trafficking of leukocytes into organ tissue. We hypothesized that the VAT and SAT microcirculations respond differently to postprandial processing of dietary fat. We also tested whether inhibition of endothelial postprandial responses to high-fat meals (HFMs) preserves metabolic health in chronic obesity. We demonstrate that administration of a single HFM or ad libitum access to a HFD for 24 h quickly induces a transient P-selectin–dependent inflammatory phenotype in the VAT but not the SAT microcirculation of lean wild-type mice. Studies in P-selectin–deficient mice confirmed a mechanistic role for P-selectin in the initiation of leukocyte trafficking, myeloperoxidase accumulation, and acute reduction in adiponectin mRNA expression by HFMs. Despite reduced VAT inflammation in response to HFMs, P-selectin–deficient mice still developed glucose intolerance and insulin resistance when chronically fed an HFD. Our data uncover a novel nutrient-sensing role of the vascular endothelium that instigates postprandial VAT inflammation. They also demonstrate that inhibition of this transient postprandial inflammatory response fails to correct metabolic dysfunction in diet-induced obesity.—Preston, K. J., Rom, I., Vrakas, C., Landesberg, G., Etwebe, Z., Muraoka, S., Autieri, M., Eguchi, S., Scalia, R. Postprandial activation of leukocyte-endothelium interaction by fatty acids in the visceral adipose tissue microcirculation.

Nitric oxide (NO) and S-nitrosothiol (SNO) are considered cardio- and vasoprotective substances. We now understand that one mechanism in which NO/SNOs provide cardiovascular protection is through their direct inhibition of cardiac G protein-coupled receptor (GPCR) kinase 2 (GRK2) activity via S-nitrosylation of GRK2 at cysteine 340 (C340). This maintains GPCR homeostasis, including β-adrenergic receptors, through curbing receptor GRK2-mediated desensitization. Previously, we have developed a knockin mouse (GRK2-C340S) where endogenous GRK2 is resistant to dynamic S-nitrosylation, which led to increased GRK2 desensitizing activity. This unchecked regulation of cardiac GRK2 activity resulted in significantly more myocardial damage after ischemic injury that was resistant to NO-mediated cardioprotection. Although young adult GRK2-C340S mice show no overt phenotype, we now report that as these mice age, they develop significant cardiovascular dysfunction due to the loss of SNO-mediated GRK2 regulation. This pathological phenotype is apparent as early as 12 mo of age and includes reduced cardiac function, increased cardiac perivascular fibrosis, and maladaptive cardiac hypertrophy, which are common maladies found in patients with cardiovascular disease (CVD). There are also vascular reactivity and aortic abnormalities present in these mice. Therefore, our data demonstrate that a chronic and global increase in GRK2 activity is sufficient to cause cardiovascular remodeling and dysfunction, likely due to GRK2’s desensitizing effects in several tissues. Because GRK2 levels have been reported to be elevated in elderly CVD patients, GRK2-C340 mice can give insight into the aged-molecular landscape leading to CVD. NEW & NOTEWORTHY Research on G protein-coupled receptor kinase 2 (GRK2) in the setting of cardiovascular aging is largely unknown despite its strong established functions in cardiovascular physiology and pathophysiology. This study uses a mouse model of chronic GRK2 overactivity to further investigate the consequences of long-term GRK2 on cardiac function and structure. We report for the first time that chronic GRK2 overactivity was able to cause cardiac dysfunction and remodeling independent of surgical intervention, highlighting the importance of GRK activity in aged-related heart disease.

Objective Mice with global null mutation of Ceacam1 (Cc1−/−), display impairment of insulin clearance that causes hyperinsulinemia followed by insulin resistance, elevated hepatic de novo lipogenesis, and visceral obesity. In addition, they manifest abnormal vascular permeability and elevated blood pressure. Liver-specific rescuing of Ceacam1 reversed all of the metabolic abnormalities in Cc1−/−liver+ mice. The current study examined whether Cc1−/− male mice develop endothelial and cardiac dysfunction and whether this relates to the metabolic abnormalities caused by defective insulin extraction. Methods and results Myography studies showed reduction of agonist-stimulated nitric oxide production in resistance arterioles in Cc1−/−, but not Cc1−/−liver+ mice. Liver-based rescuing of CEACAM1 also attenuated the abnormal endothelial adhesiveness to circulating leukocytes in parallel to reducing plasma endothelin-1 and recovering plasma nitric oxide levels. Echocardiography studies revealed increased septal wall thickness, cardiac hypertrophy and reduced cardiac performance in Cc1−/−, but not Cc1−/−xliver+ mice. Insulin signaling experiments indicated compromised IRS1/Akt/eNOS pathway leading to lower nitric oxide level, and activated Shc/MAPK pathway leading to more endothelin-1 production in the aortae and hearts of Cc1−/−, but not Cc1−/−xliver+ mice. The increase in the ratio of endothelin-1 receptor A/B indicated an imbalance in the vasomotor activity of Cc1−/− mice, which was normalized in Cc1−/−xliver+ mice. Conclusions The data underscore a critical role for impaired CEACAM1-dependent hepatic insulin clearance pathways and resulting hyperinsulinemia and lipid accumulation in aortae and heart in regulating the cardiovascular function., Highlights • Mice with global deletion of Ceacam1 gene (Cc1−/−) manifest endothelial dysfunction which is reversed by liver-specific rescuing of CEACAM1. • Restoring CEACAM1 expression in the liver reversed cardiac hypertrophy and rescued cardiac performance. • Hyperinsulinemia emerging from impaired insulin clearance regulates endothelial and cardiovascular functions.

Nitric oxide (NO) and S-nitrosothiol (SNO) have cardiovascular-protective properties and we have shown that one mechanism is through SNO-modification of G protein-coupled receptor (GPCR) kinase 2 (GRK2). S-nitrosylation at Cys340 (C340S) of GRK2 inhibits its activity on GPCRs such as β-adrenergic receptors in the heart and other GRK2 dependent processes. S-nitrosylation is believed to act as a brake on this pathological kinase. We generated global knock-in (KI) mice that express endogenous levels of GRK2, but with C340 mutated to Ser (GRK2-C340S). The C340 site is the primary site of S-nitrosylation and is largely responsible for eNOS-mediated cardioprotection after acute ischemic injury of the heart. In this study we observed that the loss of SNO-mediated inhibition of GRK2 leads to cardiovascular dysfunction during normal aging. GRK2 has been shown to be up-regulated during several cardiovascular (CV) pathologies including hypertension and heart failure, and aging is a major risk factor of CV disease (CVD). GRK2-C340S KI mice compared to their wild-type (WT) controls showed significant pathology as early as 52 weeks of age and was maintained up to 82 weeks. Maladaptive cardiac hypertrophy was present in aged GRK2-C340S mice with significant perivascular myocardial fibrosis. Fibrosis was prevalent in the kidneys as well. These histological changes were accompanied by reduced systolic function and pulse pressure compared to WT mice. Within the vasculature, aged GRK2-C340S aortas had reduced medial thickness and had reduced contraction in response to PE, KCl, or L-NAME. Acetylcholine-induced relaxation responses remained intact. Consistent with enhanced GRK2 activity in vivo in the heart, we found increased circulating levels of serum catecholamines. Many of these phenotypes we observed in GRK2-C340S mice are also observed in human aging and CVD. These phenotypes predispose patients to developing other diseases. As the US population becomes increasingly older, understanding the dynamic between aging and CVD is important in disease management and therapy. The aged GRK2-C340S mouse model represents a model of higher CVD risk and provides insights into consequences of chronic GRK2 global over-activity when the regulation of this kinase by NO is lost.

Angiogenesis is a physiological process vital for growth, development and wound healing but is also a promising therapeutic target for the treatment of ischemic cardiovascular disease. Presently, there is nothing known regarding the role of, the RNA binding protein, Interleukin Enhancer-binding Factor 3 (ILF3) in angiogenesis. RNA binding proteins play crucial roles in cellular processes, specifically, post-transcriptional control of RNAs through mRNA stabilization. We hypothesized that ILF3 plays an essential role in angiogenesis through the stabilization of pro-angiogenic mRNA transcripts. Using immunohistochemistry we identified abundant ILF3 expression in CD31 + vessels of hypoxic porcine cardiac tissue exposed to ischemic myocardial infarction. Using both western blotting and qRT-PCR, we found that pro-angiogenic stimuli, IL-19 and VEGF, induce ILF3 expression in cultured human coronary artery endothelial cells (hEC). Angiogenic proliferation, migration and tube formation are all significantly reduced in hEC when ILF3 is knocked down using siRNA. Importantly, these angiogenic assays are significantly increased when ILF3 is overexpressed in hEC using adenovirus. Using ILF3 siRNA in addition to VEGF stimulation in hEC, several angiogenic factors including CXCL1, IL-8, and HGF are decreased at the transcript and protein level. However, these angiogenic factors are increased when hEC are infected with AdILF3 and stimulated with VEGF. Through immunohistochemistry we found that ILF3 translocates from the nucleus to the cytoplasm of hEC stimulated with VEGF or IL-19 at various time points, suggesting a role in mRNA stability. Using the transcription inhibitor actinomycin D, we found that ILF3 stabilizes pro-angiogenic transcripts including VEGF, CXCL1, and IL-8 in hEC. Together these data suggest that in endothelial cells, the RNA stability protein, ILF3, plays a novel and central role in angiogenesis. We believe that ILF3 is impacting functional angiogenesis through cytokine-inducible mRNA stabilization of pro-angiogenic transcripts.

Expanding adipose depots experience hypoxia and inflammation, a phenomenon considered pathogenic of metabolic and cardiovascular complications. Currently very little is known about the potential for endogenously expressed anti-inflammatory cytokines to attenuate inflammation and also provide pro-angiogenic effects. Interleukin-19 is a uniquely anti-inflammatory, pro-angiogenic cytokine expressed in adipose tissue. We hypothesized that in the inflamed adipose depots of the overweight-obese organism IL-19 promotes a compensatory, anti-inflammatory and pro-angiogenic action to restore adipocyte homeostasis in the face of excess energy intake. We report that IL-19 is constitutively expressed in visceral and subcutaneous adipose depots of lean wild-type mice. We also found evidence of significantly increased IL-19 expression in the visceral adipose depots of diet-induced obese mice (p Il19 -/- mice, we first demonstrate that the loss of IL-19 leads to a reduction in the metabolically protective and anti-inflammatory genes PPARγ and adiponectin. Simultaneously, pro-inflammatory factors including TNFα and IL-6 are increased, and pro-angiogenic factors such as VEGF are down regulated. Il19 -/- mice display elevated fasted blood glucose levels relative to WT controls (p Il19 -/- mice exhibit insulin resistance compared to WT controls. The metabolic profile of Il19 -/- mice appears to be related to IL-19 actions on adipose tissue as suggested by the fact that Il19 -/- mice fed high fat diet experience significant down-regulation of IL-19 receptor (IL-20Rα/IL-20Rβ) expression in the visceral adipose tissue, but not in the liver and skeletal muscle. Mechanistic studies in differentiated 3T3-L1 adipocytes demonstrated that addition of rIL-19 increases PPARγ and adiponectin at both the transcript and protein level. These data suggest that potentiation of endogenous IL-19 activity may represent a novel therapeutic strategy to avert the inflammatory and metabolic complications associated with the adipose tissue dysfunction of diet-induced obesity.

MPO (myeloperoxidase) is a peroxidase enzyme secreted by activated leukocytes that plays a pathogenic role in cardiovascular disease, mainly by initiating endothelial dysfunction. The molecular mechanisms of the endothelial damaging action of MPO remain though largely elusive. Calpain is a calcium-dependent protease expressed in the vascular wall. Activation of calpains has been implicated in inflammatory disorders of the vasculature. Using endothelial cells and genetically modified mice, this study identifies the µ-calpain isoform as novel downstream signaling target of MPO in endothelial dysfunction. Mouse lung microvascular endothelial cells were stimulated with 10 nmol/L MPO for 180 minutes. MPO denitrosylated µ-calpain C-terminus domain, and time dependently activated µ-calpain, but not the m-calpain isoform. MPO also reduced Thr 172 AMPK (AMP-activated protein kinase) and Ser 1177 eNOS (endothelial nitric oxide synthase) phosphorylation via upregulation of PP2A (protein phosphatase 2) expression. At the functional level, MPO increased endothelial VCAM-1 (vascular cell adhesion molecule 1) abundance and the adhesion of leukocytes to the mouse aorta. In MPO-treated endothelial cells, pharmacological inhibition of calpain activity attenuated expression of VCAM-1 and PP2A, and restored Thr 172 AMPK and Ser 1177 eNOS phosphorylation. Compared with wild-type mice, µ-calpain deficient mice experienced reduced leukocyte adhesion to the aortic endothelium in response to MPO. Our data first establish a role for calpain in the endothelial dysfunction and vascular inflammation of MPO. The MPO/calpain/PP2A signaling pathway may provide novel pharmacological targets for the treatment of inflammatory vascular disorders.

Myeloperoxidase (MPO) is a peroxidase enzyme secreted by activated leukocytes, which has been associated with endothelial dysfunction and insulin resistance. The calcium dependent protease calpain has also been linked to vascular disease in insulin resistance and type 2 diabetes. Accordingly, we tested the hypothesis that endothelial expressed calpains play a role in MPO-induced endothelial dysfunction and vascular inflammation. Mouse lung microvascular endothelial cells (MMVEC) were stimulated with 10 nM MPO for 30, 60, 120, 180, and 240 minutes. Expression levels of Vascular Cell Adhesion Molecule 1 (VCAM-1), 5’ AMP Activated Protein Kinase (AMPK), Protein Kinase B (PKB) (Akt), endothelial Nitric Oxide (eNOS) phosphorylation at serine 1177 (Se1177), Protein Phosphatase 2 (PP2A), and calpains were measured by immunoblot analyses. MPO time dependently activated μ-calpain (Pex vivo leukocyte adhesion assay we also found leukocytes failed to adhere to the vascular endothelium of MPO treated aortas isolated from μ-calpain deficient mice (p

Genetic variants at the solute carrier family 39 member 8 (SLC39A8) gene locus are associated with the regulation of whole-blood manganese (Mn) and multiple physiological traits. SLC39A8 encodes ZIP8, a divalent metal ion transporter best known for zinc transport. Here, we hypothesized that ZIP8 regulates Mn homeostasis and Mn-dependent enzymes to influence metabolism. We generated Slc39a8-inducible global-knockout (ZIP8-iKO) and liver-specific-knockout (ZIP8-LSKO) mice and observed markedly decreased Mn levels in multiple organs and whole blood of both mouse models. By contrast, liver-specific overexpression of human ZIP8 (adeno-associated virus-ZIP8 [AAV-ZIP8]) resulted in increased tissue and whole blood Mn levels. ZIP8 expression was localized to the hepatocyte canalicular membrane, and bile Mn levels were increased in ZIP8-LSKO and decreased in AAV-ZIP8 mice. ZIP8-LSKO mice also displayed decreased liver and kidney activity of the Mn-dependent enzyme arginase. Both ZIP8-iKO and ZIP8-LSKO mice had defective protein N-glycosylation, and humans homozygous for the minor allele at the lead SLC39A8 variant showed hypogalactosylation, consistent with decreased activity of another Mn-dependent enzyme, β-1,4-galactosyltransferase. In summary, hepatic ZIP8 reclaims Mn from bile and regulates whole-body Mn homeostasis, thereby modulating the activity of Mn-dependent enzymes. This work provides a mechanistic basis for the association of SLC39A8 with whole-blood Mn, potentially linking SLC39A8 variants with other physiological traits.

The endoplasmic reticulum Ca(2+)-sensing STIM proteins mediate Ca(2+) entry signals by coupling to activate plasma membrane Orai channels. We reveal that STIM-Orai coupling is rapidly blocked by hypoxia and the ensuing decrease in cytosolic pH. In smooth muscle cells or HEK293 cells coexpressing STIM1 and Orai1, acute hypoxic conditions rapidly blocked store-operated Ca(2+) entry and the Orai1-mediated Ca(2+) release-activated Ca(2+) current (I(CRAC)). Hypoxia-induced blockade of Ca(2+) entry and I(CRAC) was reversed by NH(4)(+)-induced cytosolic alkalinization. Hypoxia and acidification both blocked I(CRAC) induced by the short STIM1 Orai-activating region. Although hypoxia induced STIM1 translocation into junctions, it did not dissociate the STIM1-Orai1 complex. However, both hypoxia and cytosolic acidosis rapidly decreased Förster resonance energy transfer (FRET) between STIM1-YFP and Orai1-CFP. Thus, although hypoxia promotes STIM1 junctional accumulation, the ensuing acidification functionally uncouples the STIM1-Orai1 complex providing an important mechanism protecting cells from Ca(2+) overload under hypoxic stress conditions.

Rationale: The cytosolic protease calpain has been recently implicated in the vascular remodeling of angiotensin II (Ang II) type 1 receptor (AT 1 R) signaling. The role of Ang II/AT 1 R/calpain signaling on endothelial function, an important and early determinant of vascular pathology, remains though totally unknown. Accordingly, we investigated the role of calpain in the endothelial dysfunction of Ang II. Objective: To demonstrate a mechanistic role for calpain in the endothelial dysfunction induced by Ang II/AT 1 R signaling. To establish endothelial-expressed calpains as an important target of AT 1 R signaling. Methods and Results: Subchronic administration of nonpressor doses of Ang II to rats and mice significantly increased vascular calpain activity via AT 1 R signaling. Intravital microscopy studies revealed that activation of vascular expressed calpains causes endothelial dysfunction with increased leukocyte–endothelium interactions and albumin permeability in the microcirculation. Western blot and immunohistochemistry studies confirmed that Ang II/AT 1 R signaling preferentially activates the constitutively expressed μ-calpain isoform and demonstrated a calpain-dependent degradation of IκBα, along with upregulation of nuclear factor κB–regulated endothelial cell adhesion molecules. These physiological and biochemical parameters were nearly normalized following inhibition of AT 1 R or calpain in vivo. RNA silencing studies in microvascular endothelial cells, along with knockout and transgenic mouse studies, further confirmed the role of μ-calpain in the endothelial adhesiveness induced by Ang II. Conclusions: This study uncovers a novel role for calpain in the endothelial dysfunction of Ang II/AT 1 R signaling and establishes the calpain system as a novel molecular target of the vascular protective action of renin–angiotensin system inhibition. Our results may have significant clinical implications in vascular disease.

Rationale: Human genetics have implicated the 5-lipoxygenase enzyme in the pathogenesis of cardiovascular disease, and an inhibitor of the 5-lipoxygenase activating protein (FLAP) is in clinical development for asthma. Objective: Here we determined whether FLAP deletion modifies the response to vascular injury. Methods and Results: Vascular remodeling was characterized 4 weeks after femoral arterial injury in FLAP knockout mice and wild-type controls. Both neointimal hyperplasia and the intima/media ratio of the injured artery were significantly reduced in the FLAP knockouts, whereas endothelial integrity was preserved. Lesional myeloid cells were depleted and vascular smooth muscle cell (VSMC) proliferation, as reflected by bromodeoxyuridine incorporation, was markedly attenuated by FLAP deletion. Inflammatory cytokine release from FLAP knockout macrophages was depressed, and their restricted ability to induce VSMC migration ex vivo was rescued with leukotriene B 4 . FLAP deletion restrained injury and attenuated upregulation of the extracellular matrix protein, tenascin C, which affords a scaffold for VSMC migration. Correspondingly, the phenotypic modulation of VSMC to a more synthetic phenotype, reflected by morphological change, loss of α-smooth muscle cell actin, and upregulation of vascular cell adhesion molecule-1 was also suppressed in FLAP knockout mice. Transplantation of FLAP-replete myeloid cells rescued the proliferative response to vascular injury. Conclusions: Expression of lesional FLAP in myeloid cells promotes leukotriene B 4 -dependent VSMC phenotypic modulation, intimal migration, and proliferation.

Prostacyclin (PGI(2)) is a vasodilator and platelet inhibitor, properties consistent with cardioprotection. More than a decade ago, inhibition of cyclooxygenase-2 (COX-2) by the nonsteroidal anti-inflammatory drugs (NSAIDs) rofecoxib and celecoxib was found to reduce the amount of the major metabolite of PGI(2) (PGI-M) in the urine of healthy volunteers. This suggested that NSAIDs might cause adverse cardiovascular events by reducing production of cardioprotective PGI(2). This prediction was based on the assumption that the concentration of PGI-M in urine likely reflected vascular production of PGI(2) and that other cardioprotective mediators, especially nitric oxide (NO), were not able to compensate for the loss of PGI(2). Subsequently, eight placebo-controlled clinical trials showed that NSAIDs that block COX-2 increase adverse cardiovascular events. We connect tissue-specific effects of NSAID action and functional correlates in mice with clinical outcomes in humans by showing that deletion of COX-2 in the mouse vasculature reduces excretion of PGI-M in urine and predisposes the animals to both hypertension and thrombosis. Furthermore, vascular disruption of COX-2 depressed expression of endothelial NO synthase and the consequent release and function of NO. Thus, suppression of PGI(2) formation resulting from deletion of vascular COX-2 is sufficient to explain the cardiovascular hazard from NSAIDs, which is likely to be augmented by secondary mechanisms such as suppression of NO production.