Spinal Stroke: Outcome Attenuation by Erythropoietin and Carbamylated Erythropoietin and Its Prediction by Sphingosine-1-Phosphate Serum Levels in Mice.

Spinal strokes may be associated with tremendous spinal cord injury. Erythropoietin (EPO) improves the neurological outcome of animals after spinal cord ischemia (SCI) and its effects on ischemia-induced endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are considered possible molecular mechanisms. Furthermore, sphingosin-1-phosphate (S1P) is suggested to correlate with SCI. In this study, the effect of recombinant human EPO (rhEPO) and carbamylated EPO (cEPO-Fc) on the outcome of mice after SCI and a prognostic value of S1P were investigated. SCI was induced in 12-month-old male mice by thoracic aortal cross-clamping after administration of rhEPO, cEPO-Fc, or a control. The locomotory behavior of mice was evaluated by the Basso mouse scale and S1P serum levels were measured by liquid chromatography-tandem mass spectrometry. The spinal cord was examined histologically and the expressions of key UPR proteins (ATF6, PERK, and IRE1a, caspase-12) were analyzed utilizing immunohistochemistry and real-time quantitative polymerase chain reaction. RhEPO and cEPO-Fc significantly improved outcomes after SCI. The expression of caspase-12 significantly increased in the control group within the first 24 h of reperfusion. Animals with better locomotory behavior had significantly higher serum levels of S1P. Our data indicate that rhEPO and cEPO-Fc have protective effects on the clinical outcome and neuronal tissue of mice after SCI and that the ER is involved in the molecular mechanisms. Moreover, serum S1P may predict the severity of impairment after SCI.

Serum Sphingosine-1-Phosphate Levels Are Associated With Severity and Outcome in Patients With Cerebral Ischemia.

BACKGROUND AND PURPOSE: The aim of this study was to examine whether sphingosine-1-phosphate (S1P) levels in patients with acute stroke are associated with stroke severity and outcome. METHODS: In a prospective stroke cohort (MARK-STROKE), 374 patients with acute ischemic stroke or transient ischemic attack were enrolled (mean age: 67.9±13.0 years, sex: 64.7% male), and serum-S1P at admission was analyzed with tandem mass spectrometry. In addition to cross-sectional analyses, 79 adverse events (death, stroke, myocardial infarction, rehospitalization) were recorded in 270 patients during follow-up. Regression analyses were adjusted for age, sex, low-density lipoprotein cholesterol, and vascular risk factors. Results were validated in an independent stroke cohort with 219 patients with acute ischemic stroke (CIRCULAS). RESULTS: Low serum-S1P was associated with higher National Institutes of Health Stroke Scale score at admission and with anterior circulation nonlacunar infarcts determined by multivariate regression analyses. During a follow-up of 294±170 days, patients with S1P in the lowest tertile (<1.33 µmol/L) had more adverse events (Kaplan-Meier analysis, P=0.048 for trend). In adjusted Cox regression analysis, the lowest S1P tertile was associated with a worse outcome after stroke (hazard ratio, HR 0.51 [95% confidence interval 0.28-0.92]). Results were confirmed in an independent cohort, ie, low S1P levels were associated with higher National Institutes of Health Stroke Scale, larger infarct volumes and worse outcome after 90 days (ß-coefficient: -0.03, P=0.026; ß-coefficient: -0.099, P=0.009 and odds ratio 0.52 [0.28-0.96], respectively). CONCLUSIONS: Our findings imply a detrimental role of low S1P levels in acute stroke and therefore underpin the therapeutic potential of S1P-mimics.

Reference ranges for sphingosine-1-phosphate in neonates.

OBJECTIVES: Sphingosine-1-phosphate (S1P) is a signalling lipid involved in embryonic development, physiological homeostasis, and pathogenic processes in multiple organ systems. Disturbance of S1P homeostasis has been associated with various human diseases in which the immune response and vascular integrity are severely compromised. Up-to-date, no study has analyzed S1P levels in neonates. The objective of this study was to determine S1P serum concentrations in neonates and establish S1P reference ranges. METHODS: S1P levels in the umbilical cord blood of 460 term and preterm neonates were compared to a previously described cohort of healthy adult blood donors. S1P levels were further correlated with demographic characteristics, cellular sources of S1P, and inflammatory markers. RESULTS: The median S1P serum level in neonates was 1.70 µmol/L (IQR 1.41-1.97 µmol/L) and significantly higher than normal values reported in adults. S1P levels correlated positively with the number of red blood cells (p<0.001) and negatively with neutrophil precursors (p=0.028). CONCLUSIONS: Elevated S1P levels in neonates compared to adults possibly result from higher S1P content in its cellular sources due to the essential role of S1P during embryogenesis. Generated S1P ranges may be used as reference ranges for future studies in neonates.

Personalised haemodynamic management targeting baseline cardiac index in high-risk patients undergoing major abdominal surgery: a randomised single-centre clinical trial.

BACKGROUND: Despite several clinical trials on haemodynamic therapy, the optimal intraoperative haemodynamic management for high-risk patients undergoing major abdominal surgery remains unclear. We tested the hypothesis that personalised haemodynamic management targeting each individual's baseline cardiac index at rest reduces postoperative morbidity. METHODS: In this single-centre trial, 188 high-risk patients undergoing major abdominal surgery were randomised to either routine management or personalised haemodynamic management requiring clinicians to maintain personal baseline cardiac index (determined at rest preoperatively) using an algorithm that guided intraoperative i.v. fluid and/or dobutamine administration. The primary outcome was a composite of major complications (European Perioperative Clinical Outcome definitions) or death within 30 days of surgery. Secondary outcomes included postoperative morbidity (assessed by a postoperative morbidity survey), hospital length of stay, mortality within 90 days of surgery, and neurocognitive function assessed after postoperative Day 3. RESULTS: The primary outcome occurred in 29.8% (28/94) of patients in the personalised management group, compared with 55.3% (52/94) of patients in the routine management group (relative risk: 0.54, 95% confidence interval [CI]: 0.38 to 0.77; absolute risk reduction: -25.5%, 95% CI: -39.2% to -11.9%; P<0.001). One patient assigned to the personalised management group, compared with five assigned to the routine management group, died within 30 days after surgery (P=0.097). There were no clinically relevant differences between the two groups for secondary outcomes. CONCLUSIONS: In high-risk patients undergoing major abdominal surgery, personalised haemodynamic management reduces a composite outcome of major postoperative complications or death within 30 days after surgery compared with routine care. CLINICAL TRIAL REGISTRATION: NCT02834377.

Sphingosine-1-phosphate receptor 1 regulates neointimal growth in a humanized model for restenosis.

OBJECTIVE: The main objective of this study was to define a role of sphingosine-1-phosphate receptor 1 (S1PR1) in the arterial injury response of a human artery. The hypotheses were tested that injury induces an expansion of S1PR1-positive cells and that these cells accumulate toward the lumen because they follow the sphingosine-1-phosphate gradient from arterial wall tissue (low) to plasma (high). METHODS: A humanized rat model was used in which denuded human internal mammary artery (IMA) was implanted into the position of the abdominal aorta of immunosuppressed Rowett nude rats. This injury model is characterized by medial as well as intimal hyperplasia, whereby intimal cells are of human origin. At 7, 14, and 28 days after implantation, grafts were harvested and processed for fluorescent immunostaining for S1PR1 and smooth muscle α-actin. Nuclei were stained with 4',6-diamidine-2'-phenylindole dihydrochloride. Using digitally reconstructed, complete cross sections of grafts, intimal and medial areas were measured, whereby the medial area had virtually been divided into an outer (toward adventitia) and inner (toward lumen) layer. The fraction of S1PR1-positive cells was determined in each layer by counting S1PR1-positive and S1PR1-negative cells. RESULTS: The fraction of S1PR1-postive cells in naive IMA is 58.9% ± 6.0% (mean ± standard deviation). At day 28 after implantation, 81.6% ± 4.4% of medial cells were scored S1PR1 positive (P < .01). At day 14, the ratio between S1PR1-positive and S1PR1-negative cells was significantly higher in the lumen-oriented inner layer (9.3 ± 2.1 vs 6.0 ± 1.0; P < .01). Cells appearing in the intima at day 7 and day 14 were almost all S1PR1 positive. At day 28, however, about one-third of intimal cells were scored S1PR1 negative. CONCLUSIONS: From these data, we conclude that denudation of IMA specifically induces the expansion of S1PR1-positive cells. Based on the nonrandom distribution of S1PR1-positive cells, we consider the possibility that much like lymphocytes, S1PR1-positive smooth muscle cells also use S1PR1 to recognize the sphingosine-1-phosphate gradient from tissue (low) to plasma (high) and so migrate out of the media toward the intima of the injured IMA.

Syndecan-1 as a biomarker for sepsis survival after major abdominal surgery.

AIM: Sepsis is a serious complication following surgery and identification of patients at risk is of high importance. Syndecan-1 (sSDC1) levels are known to be elevated during sepsis. MATERIALS & METHODS: Fifty-five patients scheduled for major abdominal surgery were prospectively included and sSDC1 concentrations were measured during hospital stay. RESULTS: Patients with postoperative sepsis showed a continued increase of sSDC1 levels and exhibited higher median sSDC1 concentrations at day 1 compared with nonseptic patients 90.3 versus 16.5 ng/ml. A significant association of sSDC1 levels with the incidence of sepsis and death was demonstrated. CONCLUSION: This study identifies sSDC1 as potential biomarker for sepsis and survival after abdominal surgery.

Clinical Molecular Imaging of Chemokine Receptor CXCR4 Expression in Atherosclerotic Plaque Using 68Ga-Pentixafor PET: Correlation with Cardiovascular Risk Factors and Calcified Plaque Burden.

The CXC-motif chemokine receptor 4 (CXCR4) represents a promising target for molecular imaging of different CXCR4-positive cell types in cardiovascular diseases such as atherosclerosis and arterial wall injury. The aim of this study was to assess the prevalence, pattern, and clinical correlates of arterial wall accumulation of 68Ga-pentixafor, a specific CXCR4 ligand for PET. Methods: The data for 51 patients who underwent 68Ga-pentixafor PET/CT for noncardiovascular indications were retrospectively analyzed. Tracer accumulation in the vessel wall of major arteries was analyzed qualitatively and semiquantitatively by blood-pool-corrected target-to-background ratios. Tracer uptake was compared with calcified plaque burden and cardiovascular risk factors. Results: Focal arterial uptake of 68Ga-pentixafor was seen at 1,411 sites in 51 (100%) of patients. 68Ga-pentixafor uptake was significantly associated with calcified plaque burden (P < 0.0001) and cardiovascular risk factors including age (P < 0.0001), arterial hypertension (P < 0.0001), hypercholesterolemia (P = 0.0005), history of smoking (P = 0.01), and prior cardiovascular events (P = 0.0004). Both the prevalence (P < 0.0001) and the signal intensity (P = 0.009) of 68Ga-pentixafor uptake increased as the number of risk factors increased. Conclusion:68Ga-pentixafor PET/CT is suitable for noninvasive, highly specific PET imaging of CXCR4 expression in the atherosclerotic arterial wall. Arterial wall 68Ga-pentixafor uptake is significantly associated with surrogate markers of atherosclerosis and is linked to the presence of cardiovascular risk factors. 68Ga-pentixafor signal is higher in patients with a high-risk profile and may hold promise for identification of vulnerable plaque.

Sphingosine-1-Phosphate: A Potential Biomarker and Therapeutic Target for Endothelial Dysfunction and Sepsis?

Sepsis is an acute life-threatening multiple organ failure caused by a dysregulated host response to infection. Endothelial dysfunction, particularly barrier disruption leading to increased vascular permeability, edema, and insufficient tissue oxygenation, is critical to sepsis pathogenesis. Sphingosine-1-phosphate (S1P) is a signaling lipid that regulates important pathophysiological processes including vascular endothelial cell permeability, inflammation, and coagulation. It is present at high concentrations in blood and lymph and at very low concentrations in tissues due to the activity of the S1P-degrading enzyme S1P-lyase in tissue cells. Recently, four preclinical observational studies determined S1P levels in serum or plasma of sepsis patients, and all found reduced S1P levels associated with the disease. Based on these findings, this review summarizes S1P-regulated processes pertaining to endothelial functions, discusses the possible use of S1P as a marker and possibilities how to manipulate S1P levels and S1P receptor activation to restore endothelial integrity, dampens the inflammatory host response, and improves organ function in sepsis.

Serum-Sphingosine-1-Phosphate Concentrations Are Inversely Associated with Atherosclerotic Diseases in Humans.

BACKGROUND AND OBJECTIVES: Atherosclerotic changes of arteries are the leading cause for deaths in cardiovascular disease and greatly impair patient's quality of life. Sphingosine-1-phosphate (S1P) is a signaling sphingolipid that regulates potentially pro-as well as anti-atherogenic processes. Here, we investigate whether serum-S1P concentrations are associated with peripheral artery disease (PAD) and carotid stenosis (CS). METHODS AND RESULTS: Serum was sampled from blood donors (controls, N = 174) and from atherosclerotic patients (N = 132) who presented to the hospital with either clinically relevant PAD (N = 102) or CS (N = 30). From all subjects, serum-S1P was measured by mass spectrometry and blood parameters were determined by routine laboratory assays. When compared to controls, atherosclerotic patients before invasive treatment to restore blood flow showed significantly lower serum-S1P levels. This difference cannot be explained by risk factors for atherosclerosis (old age, male gender, hypertension, hypercholesteremia, obesity, diabetes or smoking) or comorbidities (Chronic obstructive pulmonary disease, kidney insufficiency or arrhythmia). Receiver operating characteristic curves suggest that S1P has more power to indicate atherosclerosis (PAD and CS) than high density lipoprotein-cholesterol (HDL-C). In 35 patients, serum-S1P was measured again between one and six months after treatment. In this group, serum-S1P concentrations rose after treatment independent of whether patients had PAD or CS, or whether they underwent open or endovascular surgery. Post-treatment S1P levels were highly associated to platelet numbers measured pre-treatment. CONCLUSIONS: Our study shows that PAD and CS in humans is associated with decreased serum-S1P concentrations and that S1P may possess higher accuracy to indicate these diseases than HDL-C.

Smooth Muscle-Alpha Actin Inhibits Vascular Smooth Muscle Cell Proliferation and Migration by Inhibiting Rac1 Activity.

Smooth muscle alpha-actin (SMA) is a marker for the contractile, non-proliferative phenotype of adult smooth muscle cells (SMCs). Upon arterial injury, expression of SMA and other structural proteins decreases and SMCs acquire a pro-migratory and proliferative phenotype. To what extent SMA regulates migration and proliferation of SMCs is unclear and putative signaling pathways involved remain to be elucidated. Here, we used lentiviral-mediated gene transfer and siRNA technology to manipulate expression of SMA in carotid mouse SMCs and studied effects of SMA. Overexpression of SMA results in decreased proliferation and migration and blunts serum-induced activation of the small GTPase Rac, but not RhoA. All inhibitory effects of SMA are rescued by expression of a constitutively active Rac1 mutant (V12rac1). Moreover, reduction of SMA expression by siRNA technology results in an increased activation of Rac. Taken together, this study identifies Rac1 as a downstream target for SMA to inhibit SMC proliferation and migration.

Decreased serum concentrations of sphingosine-1-phosphate in sepsis.

INTRODUCTION: Sphingosine-1-phosphate (S1P) is a signaling lipid that regulates pathophysiological processes involved in sepsis progression, including endothelial permeability, cytokine release, and vascular tone. The aim of this study was to investigate whether serum-S1P concentrations are associated with disease severity in patients with sepsis. METHODS: This single-center prospective-observational study includes 100 patients with systemic inflammatory response syndrome (SIRS) plus infection (n = 40), severe sepsis (n = 30), or septic shock (n = 30) and 214 healthy blood donors as controls. Serum-S1P was measured by mass spectrometry. Blood parameters, including C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6), lactate, and white blood cells (WBCs), were determined by routine assays. The Sequential Organ Failure Assessment (SOFA) score was generated and used to evaluate disease severity. RESULTS: Serum-S1P concentrations were lower in patients than in controls (P < 0.01), and the greatest difference was between the control and the septic shock groups (P < 0.01). Serum-S1P levels were inversely correlated with disease severity as determined by the SOFA score (P < 0.01) as well as with IL-6, PCT, CRP, creatinine, lactate, and fluid balance. A receiver operating characteristic analysis for the presence or absence of septic shock revealed equally high sensitivity and specificity for S1P compared with the SOFA score. In a multivariate logistic regression model calculated for prediction of septic shock, S1P emerged as the strongest predictor (P < 0.001). CONCLUSIONS: In patients with sepsis, serum-S1P levels are dramatically decreased and are inversely associated with disease severity. Since S1P is a potent regulator of endothelial integrity, low S1P levels may contribute to capillary leakage, impaired tissue perfusion, and organ failure in sepsis.

Ex vivo aorta patch model for analysis of cellular adhesion.

The vascular endothelium as well as subendothelium are objects of many researches as it is directly involved in a multiplicity of physiological and pathological settings. Detailed study of endothelial function became feasible with the development of techniques to culture endothelial cells (EC) in vitro. Limitations of this approach have become apparent with the realization that cell culture dedifferentiate with time and do not exhibit properties of intact tissue. Here we describe the development of a novel ex vivo tissue model to study cell-vascular wall interactions by using isolated mouse aorta patches. Validation of this model was performed by demonstrating cell attachment and changes in cell shape typical for cell spreading during adhesion. A major advantage of this model is that cell-endothelium interaction and its molecular backgrounds can now be studied more feasibly on an intact and native tissue.

Regulator of G-protein signaling - 5 (RGS5) is a novel repressor of hedgehog signaling.

Hedgehog (Hh) signaling plays fundamental roles in morphogenesis, tissue repair, and human disease. Initiation of Hh signaling is controlled by the interaction of two multipass membrane proteins, patched (Ptc) and smoothened (Smo). Recent studies identify Smo as a G-protein coupled receptor (GPCR)-like protein that signals through large G-protein complexes which contain the Gαi subunit. We hypothesize Regulator of G-Protein Signaling (RGS) proteins, and specifically RGS5, are endogenous repressors of Hh signaling via their ability to act as GTPase activating proteins (GAPs) for GTP-bound Gαi, downstream of Smo. In support of this hypothesis, we demonstrate that RGS5 over-expression inhibits sonic hedgehog (Shh)-mediated signaling and osteogenesis in C3H10T1/2 cells. Conversely, signaling is potentiated by siRNA-mediated knock-down of RGS5 expression, but not RGS4 expression. Furthermore, using immuohistochemical analysis and co-immunoprecipitation (Co-IP), we demonstrate that RGS5 is present with Smo in primary cilia. This organelle is required for canonical Hh signaling in mammalian cells, and RGS5 is found in a physical complex with Smo in these cells. We therefore conclude that RGS5 is an endogenous regulator of Hh-mediated signaling and that RGS proteins are potential targets for novel therapeutics in Hh-mediated diseases.

VASP increases hepatic fatty acid oxidation by activating AMPK in mice.

Activation of AMP-activated protein kinase (AMPK) signaling reduces hepatic steatosis and hepatic insulin resistance; however, its regulatory mechanisms are not fully understood. In this study, we sought to determine whether vasodilator-stimulated phosphoprotein (VASP) signaling improves lipid metabolism in the liver and, if so, whether VASP's effects are mediated by AMPK. We show that disruption of VASP results in significant hepatic steatosis as a result of significant impairment of fatty acid oxidation, VLDL-triglyceride (TG) secretion, and AMPK signaling. Overexpression of VASP in hepatocytes increased AMPK phosphorylation and fatty acid oxidation and reduced hepatocyte TG accumulation; however, these responses were suppressed in the presence of an AMPK inhibitor. Restoration of AMPK phosphorylation by administration of 5-aminoimidazole-4-carboxamide riboside in Vasp(-/-) mice reduced hepatic steatosis and normalized fatty acid oxidation and VLDL-TG secretion. Activation of VASP by the phosphodiesterase-5 inhibitor, sildenafil, in db/db mice reduced hepatic steatosis and increased phosphorylated (p-)AMPK and p-acetyl CoA carboxylase. In Vasp(-/-) mice, however, sildendafil treatment did not increase p-AMPK or reduce hepatic TG content. These studies identify a role of VASP to enhance hepatic fatty acid oxidation by activating AMPK and to promote VLDL-TG secretion from the liver.

Sphingosine-1-phosphate receptor 3 promotes neointimal hyperplasia in mouse iliac-femoral arteries.

OBJECTIVE: The objective of this study was to define a role for sphingosine-1-phosphate receptor 3 (S1PR3) in intimal hyperplasia. METHODS AND RESULTS: A denudation model of the iliac-femoral artery in wild-type and S1PR3-null mice was used to define a role for S1PR3 in the arterial injury response because we found in humans and mice that expression of S1PR3 was higher in these arteries compared with carotid arteries. At 28 days after surgery, wild-type arteries formed significantly larger lesions than S1PR3-null arteries. Bromodeoxyuridine labeling experiments demonstrated that on injury, wild-type arteries exhibited higher medial as well as intimal proliferation than S1PR3-null arteries. Because S1PR3 expression in vitro was low, we expressed S1PR3 in S1PR3-null smooth muscle cells (SMCs) using retroviral-mediated gene transfer to study the effects of S1PR3 on cell functions and signaling. SMCs expressing S1PR3, but not vector-transfected controls, responded to sphingosine-1-phosphate stimulation with activation of Rac, Erk, and Akt. SMCs expressing S1PR3 also migrated more. CONCLUSIONS: In humans and mice, S1PR3 expression was higher in iliac-femoral arteries compared with carotid arteries. S1PR3 promoted neointimal hyperplasia on denudation of iliac-femoral arteries in mice, likely by stimulating cell migration and proliferation through activation of signaling pathways involving Erk, Akt, and Rac.

Reduced vascular nitric oxide-cGMP signaling contributes to adipose tissue inflammation during high-fat feeding.

OBJECTIVE: Obesity is characterized by chronic inflammation of adipose tissue, which contributes to insulin resistance and diabetes. Although nitric oxide (NO) signaling has antiinflammatory effects in the vasculature, whether reduced NO contributes to adipose tissue inflammation is unknown. We sought to determine whether (1) obesity induced by high-fat (HF) diet reduces endothelial nitric oxide signaling in adipose tissue, (2) reduced endothelial nitric oxide synthase (eNOS) signaling is sufficient to induce adipose tissue inflammation independent of diet, and (3) increased cGMP signaling can block adipose tissue inflammation induced by HF feeding. METHODS AND RESULTS: Relative to mice fed a low-fat diet, an HF diet markedly reduced phospho-eNOS and phospho-vasodilator-stimulated phosphoprotein (phospho-VASP), markers of vascular NO signaling. Expression of proinflammatory cytokines was increased in adipose tissue of eNOS-/- mice. Conversely, enhancement of signaling downstream of NO by phosphodiesterase-5 inhibition using sildenafil attenuated HF-induced proinflammatory cytokine expression and the recruitment of macrophages into adipose tissue. Finally, we implicate a role for VASP, a downstream mediator of NO-cGMP signaling in mediating eNOS-induced antiinflammatory effects because VASP-/- mice recapitulated the proinflammatory phenotype displayed by eNOS-/- mice. CONCLUSIONS: These results imply a physiological role for endothelial NO to limit obesity-associated inflammation in adipose tissue and hence identify the NO-cGMP-VASP pathway as a potential therapeutic target in the treatment of diabetes.

Endothelial NO/cGMP/VASP signaling attenuates Kupffer cell activation and hepatic insulin resistance induced by high-fat feeding.

OBJECTIVE: Proinflammatory activation of Kupffer cells is implicated in the effect of high-fat feeding to cause liver insulin resistance. We sought to determine whether reduced endothelial nitric oxide (NO) signaling contributes to the effect of high-fat feeding to increase hepatic inflammatory signaling and if so, whether this effect 1) involves activation of Kupffer cells and 2) is ameliorated by increased NO signaling. RESEARCH DESIGN AND METHODS: Effect of NO/cGMP signaling on hepatic inflammation and on isolated Kupffer cells was examined in C57BL/6 mice, eNos(-/-) mice, and Vasp(-/-) mice fed a low-fat or high-fat diet. RESULTS: We show that high-fat feeding induces proinflammatory activation of Kupffer cells in wild-type mice coincident with reduced liver endothelial nitric oxide synthase activity and NO content while, conversely, enhancement of signaling downstream of endogenous NO by phosphodiesterase-5 inhibition protects against high fat-induced inflammation in Kupffer cells. Furthermore, proinflammatory activation of Kupffer cells is evident in eNos(-/-) mice even on a low-fat diet. Targeted deletion of vasodilator-stimulated phosphoprotein (VASP), a key downstream target of endothelially derived NO, similarly predisposes to hepatic and Kupffer cell inflammation and abrogates the protective effect of NO signaling in both macrophages and hepatocytes studied in a cell culture model. CONCLUSIONS: These results collectively imply a physiological role for endothelial NO to limit obesity-associated inflammation and insulin resistance in hepatocytes and support a model in which Kupffer cell activation during high-fat feeding is dependent on reduced NO signaling. Our findings also identify the NO/VASP pathway as a novel potential target for the treatment of obesity-associated liver insulin resistance.

The adventitia: a dynamic interface containing resident progenitor cells.

Conventional views of the tunica adventitia as a poorly organized layer of vessel wall composed of fibroblasts, connective tissue, and perivascular nerves are undergoing revision. Recent studies suggest that the adventitia has properties of a stem/progenitor cell niche in the artery wall that may be poised to respond to arterial injury. It is also a major site of immune surveillance and inflammatory cell trafficking and harbors a dynamic microvasculature, the vasa vasorum, that maintains the medial layer and provides an important gateway for macrophage and leukocyte migration into the intima. In addition, the adventitia is in contact with tissue that surrounds the vessel and may actively participate in exchange of signals and cells between the vessel wall and the tissue in which it resides. This brief review highlights recent advances in our understanding of the adventitia and its resident progenitor cells and discusses progress toward an integrated view of adventitial function in vascular development, repair, and disease.

PDGF-dependent regulation of regulator of G protein signaling-5 expression and vascular smooth muscle cell functionality.

Regulator of G protein signaling (RGS) proteins, and notably members of the RGS-R4 subfamily, control vasocontractility by accelerating the inactivation of Gα-dependent signaling. RGS5 is the most highly and differently expressed RGS-R4 subfamily member in arterial smooth muscle. Expression of RGS5 first appears in pericytes during development of the afferent vascular tree, suggesting that RGS5 is a good candidate for a regulator of arterial contractility and, perhaps, for determining the mass of the smooth muscle coats required to regulate blood flow in the branches of the arterial tree. Consistent with this hypothesis, using cultured vascular smooth muscle cells (VSMCs), we demonstrate RGS5 overexpression inhibits G protein-coupled receptor (GPCR)-mediated hypertrophic responses. The next objective was to determine which physiological agonists directly control RGS5 expression in VSMCs. GPCR agonists failed to directly regulate RGS5 mRNA expression; however, platelet-derived growth factor (PDGF) acutely represses expression. Downregulation of RGS5 results in the induction of migration and the activation of the GPCR-mediated signaling pathways. This stimulation leads to the activation of mitogen-activated protein kinases directly downstream of receptor stimulation, and ultimately VSMC hypertrophy. These results demonstrate that RGS5 expression is a critical mediator of both VSMC contraction and potentially, arterial remodeling.

Reduced NO-cGMP signaling contributes to vascular inflammation and insulin resistance induced by high-fat feeding.

OBJECTIVE: Diet-induced obesity (DIO) in mice causes vascular inflammation and insulin resistance that are accompanied by decreased endothelial-derived NO production. We sought to determine whether reduced NO-cGMP signaling contributes to the deleterious effects of DIO on the vasculature and, if so, whether these effects can be blocked by increased vascular NO-cGMP signaling. METHODS AND RESULTS: By using an established endothelial cell culture model of insulin resistance, exposure to palmitate, 100 micromol/L, for 3 hours induced both cellular inflammation (activation of IKK beta-nuclear factor-kappaB) and impaired insulin signaling via the insulin receptor substrate-phosphatidylinositol 3-kinase pathway. Sensitivity to palmitate-induced endothelial inflammation and insulin resistance was increased when NO signaling was reduced using an endothelial NO synthase inhibitor, whereas endothelial responses to palmitate were blocked by pretreatment with either an NO donor or a cGMP analogue. To investigate whether endogenous NO-cGMP signaling protects against vascular responses to nutrient excess in vivo, adult male mice lacking endothelial NO synthase were studied. As predicted, both vascular inflammation (phosphorylated I kappaB alpha and intercellular adhesion molecule levels) and insulin resistance (phosphorylated Akt [pAkt] and phosphorylated eNOS [peNOS] levels) were increased in endothelial NO synthase(-/-) (eNOS(-/-)) mice, reminiscent of the effect of DIO in wild-type controls. Next, we asked whether the vascular response to DIO in wild-type mice can be reversed by a pharmacological increase of cGMP signaling. C57BL6 mice were either fed a high-fat diet or remained on a low-fat diet for 8 weeks. During the final 2 weeks of the study, mice on each diet received either placebo or the phosphodiesterase-5 inhibitor sildenafil, 10 mg/kg per day orally. In high-fat diet-fed mice, vascular inflammation and insulin resistance were completely prevented by sildenafil administration at a dose that had no effect in mice fed the low-fat diet. CONCLUSIONS: Reduced signaling via the NO-cGMP pathway is a mediator of vascular inflammation and insulin resistance during overnutrition induced by high-fat feeding. Therefore, phosphodiesterase-5, soluble guanylyl cyclase, and other molecules in the NO-cGMP pathway (eg, protein kinase G) constitute potential targets for the treatment of vascular dysfunction in the setting of obesity.