AKAP12 deficiency impairs VEGF-induced endothelial cell migration and sprouting.

AIM: Protein kinase (PK) A anchoring protein (AKAP) 12 is a scaffolding protein that anchors PKA to compartmentalize cyclic AMP signalling. This study assessed the consequences of the downregulation or deletion of AKAP12 on endothelial cell migration and angiogenesis. METHODS: The consequences of siRNA-mediated downregulation AKAP12 were studied in primary cultures of human endothelial cells as well as in endothelial cells and retinas from wild-type versus AKAP12-/- mice. Molecular interactions were investigated using a combination of immunoprecipitation and mass spectrometry. RESULTS: AKAP12 was expressed at low levels in confluent endothelial cells but its expression was increased in actively migrating cells, where it localized to lamellipodia. In the postnatal retina, AKAP12 was expressed by actively migrating tip cells at the angiogenic front, and its deletion resulted in defective extension of the vascular plexus. In migrating endothelial cells, AKAP12 was co-localized with the PKA type II-α regulatory subunit as well as multiple key regulators of actin dynamics and actin filament-based movement; including components of the Arp2/3 complex and the vasodilator-stimulated phosphoprotein (VASP). Fitting with the evidence of a physical VASP/AKAP12/PKA complex, it was possible to demonstrate that the VEGF-stimulated and PKA-dependent phosphorylation of VASP was dependent on AKAP12. Indeed, AKAP12 colocalized with phospho-Ser157 VASP at the leading edge of migrating endothelial cells. CONCLUSION: The results suggest that compartmentalized AKAP12/PKA signalling mediates VASP phosphorylation at the leading edge of migrating endothelial cells to translate angiogenic stimuli into altered actin dynamics and cell movement.

Nitric oxide maintains endothelial redox homeostasis through PKM2 inhibition.

Decreased nitric oxide (NO) bioavailability and oxidative stress are hallmarks of endothelial dysfunction and cardiovascular diseases. Although numerous proteins are S-nitrosated, whether and how changes in protein S-nitrosation influence endothelial function under pathophysiological conditions remains unknown. We report that active endothelial NO synthase (eNOS) interacts with and S-nitrosates pyruvate kinase M2 (PKM2), which reduces PKM2 activity. PKM2 inhibition increases substrate flux through the pentose phosphate pathway to generate reducing equivalents (NADPH and GSH) and protect against oxidative stress. In mice, the Tyr656 to Phe mutation renders eNOS insensitive to inactivation by oxidative stress and prevents the decrease in PKM2 S-nitrosation and reducing equivalents, thereby delaying cardiovascular disease development. These findings highlight a novel mechanism linking NO bioavailability to antioxidant responses in endothelial cells through S-nitrosation and inhibition of PKM2.

Diagnostic error as a result of drug-laboratory test interactions.

Background Knowledge of possible drug-laboratory test interactions (DLTIs) is important for the interpretation of laboratory test results. Test results may be affected by physiological or analytical drug effects. Failure to recognize these interactions may lead to misinterpretation of test results, a delayed or erroneous diagnosis or unnecessary extra tests or therapy, which may harm patients. Content Thousands of interactions have been reported in the literature, but are often fragmentarily described and some papers even reported contradictory findings. How can healthcare professionals become aware of all these possible interactions in their individual patients? DLTI decision support applications could be a good solution. In a literature search, only four relevant studies have been found on DLTI decision support applications in clinical practice. These studies show a potential benefit of automated DLTI messages to physicians for the interpretation of laboratory test results. All physicians reported that part of the DLTI messages were useful. In one study, 74% of physicians even sometimes refrained from further additional examination. Summary and outlook Unrecognized DLTIs potentially cause diagnostic errors in a large number of patients. Therefore, efforts to avoid these errors, for example with a DLTI decision support application, could tremendously improve patient outcome.

Impact of interactions between drugs and laboratory test results on diagnostic test interpretation - a systematic review.

Intake of drugs may influence the interpretation of laboratory test results. Knowledge and correct interpretation of possible drug-laboratory test interactions (DLTIs) is important for physicians, pharmacists and laboratory specialists. Laboratory results may be affected by analytical or physiological effects of medication. Failure to take into account the possible unintended influence of drug use on a laboratory test result may lead to incorrect diagnosis, incorrect treatment and unnecessary follow-up. The aim of this review is to give an overview of the literature investigating the clinical impact and use of DLTI decision support systems on laboratory test interpretation. Particular interactions were reported in a large number of articles, but they were fragmentarily described and some papers even reported contradictory findings. To provide an overview of information that clinicians and laboratory staff need to interpret test results, DLTI databases have been made by several groups. In a literature search, only four relevant studies have been found on DLTI decision support applications for laboratory test interpretation in clinical practice. These studies show a potential benefit of automated DLTI messages to physicians for the correct interpretation of laboratory test results. Physicians reported 30-100% usefulness of DLTI messages. In one study 74% of physicians sometimes even refrained from further additional examination. The benefit of decision support increases when a refined set of clinical rules is determined in cooperation with health care professionals. The prevalence of DLTIs is high in a broad range of combinations of laboratory tests and drugs and these frequently remain unrecognized.

Endothelial AMP-Activated Kinase α1 Phosphorylates eNOS on Thr495 and Decreases Endothelial NO Formation.

AMP-activated protein kinase (AMPK) is frequently reported to phosphorylate Ser1177 of the endothelial nitric-oxide synthase (eNOS), and therefore, is linked with a relaxing effect. However, previous studies failed to consistently demonstrate a major role for AMPK on eNOS-dependent relaxation. As AMPK also phosphorylates eNOS on the inhibitory Thr495 site, this study aimed to determine the role of AMPKα1 and α2 subunits in the regulation of NO-mediated vascular relaxation. Vascular reactivity to phenylephrine and acetylcholine was assessed in aortic and carotid artery segments from mice with global (AMPKα-/-) or endothelial-specific deletion (AMPKαΔEC) of the AMPKα subunits. In control and AMPKα1-depleted human umbilical vein endothelial cells, eNOS phosphorylation on Ser1177 and Thr495 was assessed after AMPK activation with thiopental or ionomycin. Global deletion of the AMPKα1 or α2 subunit in mice did not affect vascular reactivity. The endothelial-specific deletion of the AMPKα1 subunit attenuated phenylephrine-mediated contraction in an eNOS- and endothelium-dependent manner. In in vitro studies, activation of AMPK did not alter the phosphorylation of eNOS on Ser1177, but increased its phosphorylation on Thr495. Depletion of AMPKα1 in cultured human endothelial cells decreased Thr495 phosphorylation without affecting Ser1177 phosphorylation. The results of this study indicate that AMPKα1 targets the inhibitory phosphorylation Thr495 site in the calmodulin-binding domain of eNOS to attenuate basal NO production and phenylephrine-induced vasoconstriction.

The role of eNOS on the compensatory regulation of vascular tonus by H2S in mouse carotid arteries.

The gasotransmitter nitric oxide (NO) has an important role in vascular function and a decrease in its bioavailability is accepted as a main pathological mechanism for cardiovascular diseases. However, other gasotransmitters such as hydrogen sulfide (H2S) are also generated by the endothelium and can also affect vascular tone and a crosstalk may exist between H2S and NO. We therefore investigated the consequences of deficiency, replacement or overexpression of endothelial nitric oxide synthase (eNOS) on H2S-induced vascular responses in murine carotid arteries. In pre-contracted carotid arteries from wild-type (WT) mice, l-cysteine elicited relaxation that was inhibited by the H2S synthesis inhibitor amino-oxyacetic acid (AOAA). Genetic deletion of eNOS increased l-cysteine-induced relaxation compared to WT, but the replacement of eNOS by adenoviral transfection or H2S synthesis inhibition by AOAA reversed it. Furthermore, eNOS deletion did not alter NaHS-induced relaxation in carotid arteries while eNOS overexpression/replacement increased NaHS-induced relaxation responses in carotid arteries from WT or eNOS-/-. We suggest that, endogenously produced H2S can compensate for impaired vasodilatory responses in the absence of NO to maintain vascular patency; while, eNOS abundance can limit endogenous H2S-induced vascular responses in mice carotid arteries. Our result suggests that endogenous vs. exogenous H2S-induced relaxation are reciprocally regulated by NO in mice carotid arteries.

Clinical evaluation of eight different D-dimer tests for the exclusion of deep venous thrombosis in primary care patients.

D-dimer tests are an essential element in the diagnostic work-up of deep venous thrombosis (DVT). However, the poor standardization amongst assays necessitates clinical validation before implementation in daily practice. We therefore evaluated the analytical and diagnostic performance of eight D-dimer tests in a representative group of 290 prospectively identified consecutive primary care patients with suspected DVT. Seven quantitative D-dimer assays, and a qualitative test, Simplify, were evaluated. Correlation between assays was generally poor and several assays showed a significant bias in the method comparison. Nevertheless, the Vidas D-dimer, Innovance D-dimer (CA1500 and BCS), Pathfast D-dimer, and HemosIL HS500 (ACL TOP), all displayed 100% (95% CI: 85-100%) sensitivity. Tina-quant (Modular), AQT90 D-dimer, and Liatest (STA(®)) D-dimer tests showed a slightly lower sensitivity of 95% (78-100%). and the Simplify test reached a sensitivity of 91% (72-99%) that was further improved in combination with a clinical decision rule to 95% (76-100%). In concert with the low (8.2%) prevalence of proximal DVT, diagnosed by compression ultrasonography, in our study, all test reached a negative predictive value (NPV) of at least 99%. The user friendliness of the assays differed mainly by stability of reagents, calibration frequency, time required to obtain a test result and costs of a test. In conclusion, despite considerable analytical differences, in our low-risk population all tests evaluated displayed an excellent NPV. In combination with a validated clinical decision rule to identify low-risk patients, even a straightforward POC solution could safely and cost-efficiently rule out DVT.

The number of cardiac myocytes in the hypertrophic and hypotrophic left ventricle of the obese and calorie-restricted mouse heart.

Changes in body mass due to varying amounts of calorie intake occur frequently with obesity and anorexia/cachexia being at opposite sides of the scale. Here, we tested whether a high-fat diet or calorie restriction (CR) decreases the number of cardiac myocytes and affects their volume. Ten 6-8-week-old mice were randomly assigned to a normal (control group, n = 5) or high-fat diet (obesity group, n = 5) for 28 weeks. Ten 8-week-old mice were randomly assigned to a normal (control group, n = 5) or CR diet (CR group, n = 5) for 7 days. The left ventricles of the hearts were prepared for light and electron microscopy, and analysed by design-based stereology. In CR, neither the number of cardiac myocytes, the relationship between one- and multinucleate myocytes nor their mean volume were significantly different between the groups. In contrast, in the obese mice we observed a significant increase in cell size combined with a lower number of cardiomyocytes (P < 0.05 in the one-sided U-test) and an increase in the mean number of nuclei per myocyte. The mean volume of myofibrils and mitochondria per cardiac myocyte reflected the hypertrophic and hypotrophic remodelling in obesity and CR, respectively, but were only significant in the obese mice, indicating a more profound effect of the obesity protocol than in the CR experiments. Taken together, our data indicate that long-lasting obesity is associated with a loss of cardiomyocytes of the left ventricle, but that short-term CR does not alter the number of cardiomyocytes.

Monoamine oxidases are mediators of endothelial dysfunction in the mouse aorta.

Monoamine oxidases (MAOs) generate H(2)O(2) as a by-product of their catalytic cycle. Whether MAOs are mediators of endothelial dysfunction is unknown and was determined here in the angiotensin II and lipopolysaccharide-models of vascular dysfunction in mice. Quantitative real-time polymerase chain reaction revealed that mouse aortas contain enzymes involved in catecholamine generation and MAO-A and MAO-B mRNA. MAO-A and -B proteins could be detected by Western blot not only in mouse aortas but also in human umbilical vein endothelial cells. Ex vivo incubation of mouse aorta with recombinant MAO-A increased H(2)O(2) formation and induced endothelial dysfunction that was attenuated by polyethylene glycol-catalase and MAO inhibitors. In vivo lipopolysaccharide (8 mg/kg IP overnight) or angiotensin II (1 mg/kg per day, 2 weeks, minipump) treatment induced vascular MAO-A and -B expressions and resulted in attenuated endothelium-dependent relaxation of the aorta in response to acetylcholine. MAO inhibitors reduced the lipopolysaccharide- and angiotensin II-induced aortic reactive oxygen species formation by 50% (ferrous oxidation xylenol orange assay) and partially normalized endothelium-dependent relaxation. MAO-A and MAO-B inhibitors had an additive effect; combined application completely restored endothelium-dependent relaxation. To determine how MAO-dependent H(2)O(2) formation induces endothelial dysfunction, cyclic GMP was measured. Histamine stimulation of human umbilical vein endothelial cells to activate endothelial NO synthase resulted in an increase in cyclic GMP, which was almost abrogated by MAO-A exposure. MAO inhibition prevented this effect, suggesting that MAO-induced H(2)O(2) formation is sufficient to attenuate endothelial NO release. Thus, MAO-A and MAO-B are both expressed in the mouse aorta, induced by in vivo lipopolysaccharide and angiotensin II treatment and contribute via the generation of H(2)O(2) to endothelial dysfunction in vascular disease models.

Ca2+-sensing receptor cleavage by calpain partially accounts for altered vascular reactivity in mice fed a high-fat diet.

The Ca-sensing receptor (CaSR) is expressed in endothelial and smooth muscle cells, but its role in regulating vascular reactivity is unclear, as are the effects of disease on CaSR function and expression. We studied vascular reactivity in aortic segments from healthy and diabetic mice, combined with in vitro proteolysis studies and Western blot analyses of CaSR expression in tissue samples. In endothelium-intact aortic rings, extracellular Ca elicited a nitric oxide-dependent relaxation that was attenuated by the CaSR antagonist, NPS2390. The calcimimetic, calindol, induced the endothelium-independent relaxation of aortic segments that was also sensitive to NPS2390. The antagonist failed to affect responses to acetylcholine or U46619 but attenuated contractions to phenylephrine and potassium. In mice fed a Western-type diet, phenylephrine-induced contractions and calindol-induced relaxations were markedly attenuated, and CaSR expression was decreased. The latter phenomenon could be attributed to the activation of the Ca-dependent protease, µ-calpain, and the subsequent proteolytic cleavage of the CaSR. CaSR activation in smooth muscle cells modulates vascular responsiveness to Ca-elevating agonists. These effects are blunted during metabolic stress because of the limited proteolysis of the CaSR by calpain. The loss of the CaSR function may predispose to the macrovascular late complications associated with diabetes.

11,12-EET stimulates the association of BK channel α and ß(1) subunits in mitochondria to induce pulmonary vasoconstriction.

In the systemic circulation, 11,12-epoxyeicosatrienoic acid (11,12-EET) elicits nitric oxide (NO)- and prostacyclin-independent vascular relaxation, partially through the activation of large conductance Ca(2+)-activated potassium (BK) channels. However, in the lung 11,12-EET contributes to hypoxia-induced pulmonary vasoconstriction. Since pulmonary artery smooth muscle cells also express BK channels, we assessed the consequences of BKß(1) subunit deletion on pulmonary responsiveness to 11,12-EET as well as to acute hypoxia. In buffer-perfused mouse lungs, hypoxia increased pulmonary artery pressure and this was significantly enhanced in the presence of NO synthase (NOS) and cyclooxygenase (COX) inhibitors. Under these conditions the elevation of tissue EET levels using an inhibitor of the soluble epoxide hydrolase (sEH-I), further increased the hypoxic contraction. Direct administration of 11,12-EET also increased pulmonary artery pressure, and both the sEH-I and 11,12-EET effects were prevented by iberiotoxin and absent in BKß(1)(-/-) mice. In pulmonary artery smooth muscle cells treated with NOS and COX inhibitors and loaded with the potentiometric dye, di-8-ANEPPS, 11,12-EET induced depolarization while the BK channel opener NS1619 elicited hyperpolarization indicating there was no effect of the EET on classical plasma membrane BK channels. In pulmonary artery smooth muscle cells a subpopulation of BK channels is localized in mitochondria. In these cells, 11,12-EET elicited an iberiotoxin-sensitive loss of mitochondrial membrane potential (JC-1 fluorescence) leading to plasma membrane depolarization, an effect not observed in BKß(1)(-/-) cells. Mechanistically, stimulation with 11,12-EET time-dependently induced the association of the BK α and ß(1) subunits. Our data indicate that in the absence of NO and prostacyclin 11,12-EET contributes to pulmonary vasoconstriction by stimulating the association of the α and ß(1) subunits of mitochondrial BK channels. The 11,12-EET-induced activation of BK channels results in loss of the mitochondrial membrane potential and depolarization of the pulmonary artery smooth muscle cells.

Nucleotide excision DNA repair is associated with age-related vascular dysfunction.

BACKGROUND: Vascular dysfunction in atherosclerosis and diabetes mellitus, as observed in the aging population of developed societies, is associated with vascular DNA damage and cell senescence. We hypothesized that cumulative DNA damage during aging contributes to vascular dysfunction. METHODS AND RESULTS: In mice with genomic instability resulting from the defective nucleotide excision repair genes ERCC1 and XPD (Ercc1(d/-) and Xpd(TTD) mice), we explored age-dependent vascular function compared with that in wild-type mice. Ercc1(d/-) mice showed increased vascular cell senescence, accelerated development of vasodilator dysfunction, increased vascular stiffness, and elevated blood pressure at a very young age. The vasodilator dysfunction was due to decreased endothelial nitric oxide synthase levels and impaired smooth muscle cell function, which involved phosphodiesterase activity. Similar to Ercc1(d/-) mice, age-related endothelium-dependent vasodilator dysfunction in Xpd(TTD) animals was increased. To investigate the implications for human vascular disease, we explored associations between single-nucleotide polymorphisms of selected nucleotide excision repair genes and arterial stiffness within the AortaGen Consortium and found a significant association of a single-nucleotide polymorphism (rs2029298) in the putative promoter region of DDB2 gene with carotid-femoral pulse wave velocity. CONCLUSIONS: Mice with genomic instability recapitulate age-dependent vascular dysfunction as observed in animal models and in humans but with an accelerated progression compared with wild-type mice. In addition, we found associations between variations in human DNA repair genes and markers for vascular stiffness, which is associated with aging. Our study supports the concept that genomic instability contributes importantly to the development of cardiovascular disease.

Leptin potentiates endothelium-dependent relaxation by inducing endothelial expression of neuronal NO synthase.

OBJECTIVE: Obesity is associated with hyperleptinemia but it is not clear whether leptin protects vascular function or promotes dysfunction. We therefore studied the consequences of hyperleptinemia in lean mice. METHODS AND RESULTS: Wild-type and endothelial NO synthase (eNOS)(-/-) mice were infused with leptin (0.4 mg/kg per day, 7 days), and endothelium-dependent relaxation was studied in aortic segments. Leptin had no effect on acetylcholine-induced endothelium-dependent relaxation in normal wild-type mice but restored endothelium-dependent relaxation in wild-type mice treated with angiotensin II (0.7 mg/kg per day, 7 days) to induce endothelial dysfunction. Leptin also sensitized aortae from eNOS(-/-) mice to acetylcholine, an effect blocked by neuronal NOS (nNOS) inhibition and not observed in eNOS-nNOS double(-/-) mice. Consistent with these findings, leptin induced nNOS expression in murine and human vessels and human endothelial but not smooth muscle cells. Aortic nNOS expression was also induced in mice by a high-fat diet. Mechanistically, leptin increased endothelial Janus kinase 2 and signal transducer and activator of transcription 3 phosphorylation, and inhibition of Janus kinase 2 prevented nNOS induction in cultured cells and leptin-induced relaxations in eNOS(-/-) mice. CONCLUSIONS: Leptin induces endothelial nNOS expression, which compensates, in part, for a lack of NO production by eNOS to maintain endothelium-dependent relaxation.

Stereological characterization of left ventricular cardiomyocytes, capillaries, and innervation in the nondiabetic, obese mouse.

BACKGROUND: Obesity is associated with left ventricular hypertrophy and dysfunction, but little is known about the structural remodeling of cardiomyocytes, capillaries, and nerve fibers in this state. We hypothesized that all three compartments should show quantitative structural alterations. METHODS: Ten C57Bl6 mice were randomly assigned to a control or obesity group. Lean mice received standard chow, whereas obese mice received a high-fat Western diet. After 28 weeks, the mice were sacrificed, and the hearts were prepared for design-based stereology using light and electron microscopy. RESULTS: Body mass and left ventricular mass were significantly elevated in obese vs. control mice. The left ventricular hypertrophy was accompanied by a significant increase in cardiomyocyte lipid droplets and total myocyte volume. The volume fractions of myofibrils, free sarcoplasm, and mitochondria did not differ between the groups. The total length of capillaries was significantly enhanced in obese vs. control mice, whereas the total length of axons ramifying between cardiomyocytes was not different. CONCLUSIONS: Obesity is associated with significant structural alterations in cardiomyocytes and capillaries, whereas no structural changes in the myocardial innervation were observed. The structural characteristics in obese mice do not provide a clear basis for functional changes observed in obesity-related cardiac hypertrophy.

Cytochrome P450-derived epoxyeicosatrienoic acids and pulmonary hypertension: central role of transient receptor potential C6 channels.

Hypoxia induces the constriction of pulmonary resistance arteries, which results in the redistribution of blood from poor to better ventilated areas, thus optimizing its oxygenation. Many different oxygen-sensing mechanisms have been proposed to regulate this process, including cytochrome P450 enzymes. These enzymes, which convert substrates such as arachidonic acid into bioactive epoxides (the epoxyeicosatrienoic acids [EETs]), are highly expressed in the lung as is the soluble epoxide hydrolase which metabolizes the epoxides to their less active diols. The EETs play a well-documented role as endothelium-derived vasodilators in the systemic vasculature, but in the pulmonary circulation, they are generated in vascular smooth muscle cells and potentiate vasoconstriction. Preventing the breakdown of 11,12-EET by the inhibition or genetic deletion of the soluble epoxide hydrolase strongly augments the response to hypoxia. Mechanistically, 11,12-EET potentiates the contractile response by recruiting transient receptor potential C6 channels to caveolae. Indeed, neither 11,12-EET nor hypoxia is able to elicit pulmonary vasoconstriction in TRPC6 knockout mice. The cytochrome and soluble epoxide hydrolase enzymes are also implicated in the vascular remodeling associated with chronic hypoxia and pulmonary hypertension. Thus, targeting this pathway may be in an attractive new therapeutic approach to treat this incapacitating disease.

Angiotensin II impairs endothelial function via tyrosine phosphorylation of the endothelial nitric oxide synthase.

Proline-rich tyrosine kinase 2 (PYK2) can be activated by angiotensin II (Ang II) and reactive oxygen species. We report that in endothelial cells, Ang II enhances the tyrosine phosphorylation of endothelial NO synthase (eNOS) in an AT(1)-, H(2)O(2)-, and PYK2-dependent manner. Low concentrations (1-100 micromol/liter) of H(2)O(2) stimulated the phosphorylation of eNOS Tyr657 without affecting that of Ser1177, and attenuated basal and agonist-induced NO production. In isolated mouse aortae, 30 micromol/liter H(2)O(2) induced phosphorylation of eNOS on Tyr657 and impaired acetylcholine-induced relaxation. Endothelial overexpression of a dominant-negative PYK2 mutant protected against H(2)O(2)-induced endothelial dysfunction. Correspondingly, carotid arteries from eNOS(-/-) mice overexpressing the nonphosphorylatable eNOS Y657F mutant were also protected against H(2)O(2). In vivo, 3 wk of treatment with Ang II considerably increased levels of Tyr657-phosphorylated eNOS in the aortae of wild-type but not Nox2(y/-) mice, and this was again associated with a clear impairment in endothelium-dependent vasodilatation in the wild-type but not in the Nox2(y/-) mice. Collectively, endothelial PYK2 activation by Ang II and H(2)O(2) causes the phosphorylation of eNOS on Tyr657, attenuating NO production and endothelium-dependent vasodilatation. This mechanism may contribute to the endothelial dysfunction observed in cardiovascular diseases associated with increased activity of the renin-angiotensin system and elevated redox stress.

Role of cytochrome P450-dependent transient receptor potential V4 activation in flow-induced vasodilatation.

AIMS: Fluid shear stress elicits endothelium-dependent vasodilatation via nitric oxide and prostacyclin-dependent and -independent mechanisms. The latter includes the opening of Ca(2+)-operated potassium channels by cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) leading to endothelial hyperpolarization. We previously reported that EETs activate the transient receptor potential (TRP) V4 channel in vascular endothelial cells and that Ca(2+) influx in these cells in response to mechanical stimuli is dependent on the activation of CYP epoxygenases. We therefore hypothesized that the TRPV4 channel is involved in the flow-induced vasodilatation attributed to the endothelium-derived hyperpolarizing factor (EDHF). METHODS AND RESULTS: In the presence of N(omega)-nitro-l-arginine methyl ester and diclofenac, precontracted mouse carotid arteries displayed a considerable vasodilatation in response to step-wise increases in luminal flow. The EDHF-mediated, flow-induced vasodilatation could be inhibited by the epoxygenase inhibitor MS-PPOH, was abolished after down-regulation of CYP epoxygenases in tissue culture, and could be restored by viral expression of CYP2C9 in the endothelium. The TRPV4-channel inhibitor ruthenium red (RuR) inhibited the EDHF-mediated flow response, but the combination of MS-PPOH and RuR had no further effect. RuR also inhibited the response in CYP2C9-overexpressing vessels. Moreover, TRPV4-deficient mice demonstrated a blunted EDHF-mediated response to increases in luminal flow in comparison to their wild-type littermates, and the addition of MS-PPOH was without effect in these mice (up to 38 +/- 3% in TRPV4(-/-) vs. 57 +/- 6% in TRPV4(+/+), P < 0.01). In cultured human endothelial cells, exposure to fluid shear stress induced the translocation of the TRPV4 channel from a perinuclear localization to the cell membrane. CONCLUSION: We conclude that the TRPV4 channel is involved in flow-induced, endothelium-dependent vasodilatation of murine carotid arteries. Moreover, the activation of the TRPV4 channel by flow requires an active CYP epoxygenase and the translocation of the channel to the cell membrane.

Inhibition of endothelial nitric oxide synthase activity by proline-rich tyrosine kinase 2 in response to fluid shear stress and insulin.

In native and primary cultures of endothelial cells, fluid shear stress elicits the tyrosine phosphorylation of the endothelial NO synthase (eNOS), however, the consequences of this modification on enzyme activity are unclear. We found that fluid shear stress induces the association of eNOS with the proline-rich tyrosine kinase 2 (PYK2) in endothelial cells and that the eNOS immunoprecipitated from eNOS- and PYK2-overexpressing HEK293 cells was tyrosine-phosphorylated on Tyr657. In mouse carotid arteries, the overexpression of wild-type PYK2, but not a dominant-negative PYK2, decreased eNOS activity (approximately 50%), whereas in murine lung endothelial cells, the downregulation of PYK2 (small interfering RNA) increased ionomycin-induced NO production. Mutation of Tyr657 to the phosphomimetic residues aspartate (D) or glutamate (E) abolished enzyme activity, whereas a nonphosphorylatable mutant (phenylalanine [F]) showed activity comparable to the wild-type enzyme. Moreover, normal flow-induced vasodilatation was apparent in carotid arteries from eNOS(-/-) mice overexpressing either the wild-type eNOS or the Y657F mutant, whereas no flow-induced vasodilatation was apparent in arteries expressing the Y657E eNOS mutant. Insulin also activated PYK2 and stimulated eNOS in endothelial cells expressing the Y657F mutant but not wild-type eNOS. These data indicate that PYK2 mediates the tyrosine phosphorylation of eNOS on Tyr657 in response to fluid shear stress and insulin stimulation and that this modification attenuates the activity of the enzyme. The PYK2-dependent inhibition of NO production may serve to keep eNOS activity low and limit the detrimental consequences of maintained high NO output, ie, the generation of peroxynitrite.