Endothelial CD40 Mediates Microvascular von Willebrand Factor-Dependent Platelet Adhesion Inducing Inflammatory Venothrombosis in ADAMTS13 Knockout Mice.

BACKGROUND: von Willebrand factor (vWF) plays an important role in platelet activation. CD40-CD40 ligand (CD40L) induced vWF release has been described in large vessels and cultured endothelium, but its role in the microcirculation is not known. Here, we studied whether CD40 is expressed in murine microvessels in vivo, whether CD40L induces platelet adhesion and leukocyte activation, and how deficiency of the vWF cleaving enzyme ADAMTS13 affects these processes. METHODS AND RESULTS: The role of CD40L in the formation of beaded platelet strings reflecting their adhesion to ultralarge vWF fibers (ULVWF) was analyzed in the murine cremaster microcirculation in vivo. Expression of CD40 and vWF was studied by immunohistochemistry in isolated and fixed cremasters. Microvascular CD40 was only expressed under inflammatory conditions and exclusively in venous endothelium. We demonstrate that CD40L treatment augmented the number of platelet strings, reflecting ULVWF multimer formation exclusively in venules and small veins. In ADAMTS13 knockout mice, the number of platelet strings further increased to a significant extent. As a consequence extensive thrombus formation was induced in venules of ADAMTS13 knockout mice. In addition, circulating leukocytes showed primary and rapid adherence to these platelet strings followed by preferential extravasation in these areas. CONCLUSION: CD40L is an important stimulus of microvascular endothelial ULVWF release, subsequent platelet string formation and leukocyte extravasation but only in venous vessels under inflammatory conditions. Here, the lack of ADAMTS13 leads to severe thrombus formation. The results identify CD40 expression and ADAMTS13 activity as important targets to prevent microvascular inflammatory thrombosis.

The Phosphatase SHP-2 Activates HIF-1α in Wounds In Vivo by Inhibition of 26S Proteasome Activity.

Vascular remodeling and angiogenesis are required to improve the perfusion of ischemic tissues. The hypoxic environment, induced by ischemia, is a potent stimulus for hypoxia inducible factor 1α (HIF-1α) upregulation and activation, which induce pro-angiogenic gene expression. We previously showed that the tyrosine phosphatase SHP-2 drives hypoxia mediated HIF-1α upregulation via inhibition of the proteasomal pathway, resulting in revascularization of wounds in vivo. However, it is still unknown if SHP-2 mediates HIF-1α upregulation by affecting 26S proteasome activity and how the proteasome is regulated upon hypoxia. Using a reporter construct containing the oxygen-dependent degradation (ODD) domain of HIF-1α and a fluorogenic proteasome substrate in combination with SHP-2 mutant constructs, we show that SHP-2 inhibits the 26S proteasome activity in endothelial cells under hypoxic conditions in vitro via Src kinase/p38 mitogen-activated protein kinase (MAPK) signalling. Moreover, the simultaneous expression of constitutively active SHP-2 (E76A) and inactive SHP-2 (CS) in separate hypoxic wounds in the mice dorsal skin fold chamber by localized magnetic nanoparticle-assisted lentiviral transduction showed specific regulation of proteasome activity in vivo. Thus, we identified a new additional mechanism of SHP-2 mediated HIF-1α upregulation and proteasome activity, being functionally important for revascularization of wounds in vivo. SHP-2 may therefore constitute a potential novel therapeutic target for the induction of angiogenesis in ischemic vascular disease.

PKA negatively modulates the migration enhancing effect of Connexin 43.

Connexin 43 (Cx43) expression is associated with an increased cell migration and related changes of the actin cytoskeleton (enhanced filopodia formation). These effects are mediated by the C-terminal cytoplasmic part of Cx43 in a channel-independent manner. Since this part has been shown to interact with a variety of proteins and has multiple phosphorylation sites we analyzed here a potential role of the protein kinase A (PKA) for the Cx43 mediated increase in cell migration. Mutation of the PKA-phosphorylation site (substitution of three serines by alanine or glycine) resulted in a further increase in cell motility compared to wild-type Cx43, but with a loss of directionality. Likewise, cell motility was enhanced by PKA inhibition only in Cx43 expressing cells, while reduced in the presence of the PKA activator forskolin. In contrast, cell motility remained unaffected by stimulation with forskolin in cells expressing Cx43 with the mutated PKA phosphorylation site (Cx43-PKA) as well as in Cx-deficient cells. Moreover, PKA activation resulted in increased binding of PKA and VASP to Cx43 associated with an enhanced phosphorylation of VASP, an important regulatory protein of cell polarity and directed migration. Functionally, we could confirm these results in endothelial cells endogenously expressing Cx43. A Tat-Cx43 peptide containing the PKA phosphorylation site abolished the PKA dependent reduction in endothelial cell migration. Our results indicate that PKA dependent phosphorylation of Cx43 modulates cell motility and plays a pivotal role in regulating directed cell migration.

Role of CD40 and ADAMTS13 in von Willebrand factor-mediated endothelial cell-platelet-monocyte interaction.

Monocyte extravasation into the vessel wall is a key step in atherogenesis. It is still elusive how monocytes transmigrate through the endothelial cell (EC) monolayer at atherosclerosis predilection sites. Platelets tethered to ultra-large von Willebrand factor (ULVWF) multimers deposited on the luminal EC surface following CD40 ligand (CD154) stimulation may facilitate monocyte diapedesis. Human ECs grown in a parallel plate flow chamber for live-cell imaging or Transwell permeable supports for transmigration assay were exposed to fluid or orbital shear stress and CD154. Human isolated platelets and/or monocytes were superfused over or added on top of the EC monolayer. Plasma levels and activity of the ULVWF multimer-cleaving protease ADAMTS13 were compared between coronary artery disease (CAD) patients and controls and were verified by the bioassay. Two-photon intravital microscopy was performed to monitor CD154-dependent leukocyte recruitment in the cremaster microcirculation of ADAMTS13-deficient versus wild-type mice. CD154-induced ULVWF multimer-platelet string formation on the EC surface trapped monocytes and facilitated transmigration through the EC monolayer despite high shear stress. Two-photon intravital microscopy revealed CD154-induced ULVWF multimer-platelet string formation preferentially in venules, due to strong EC expression of CD40, causing prominent downstream leukocyte extravasation. Plasma ADAMTS13 abundance and activity were significantly reduced in CAD patients and strongly facilitated both ULVWF multimer-platelet string formation and monocyte trapping in vitro. Moderate ADAMTS13 deficiency in CAD patients augments CD154-mediated deposition of platelet-decorated ULVWF multimers on the luminal EC surface, reinforcing the trapping of circulating monocytes at atherosclerosis predilection sites and promoting their diapedesis.

HIF-1α Dependent Wound Healing Angiogenesis In Vivo Can Be Controlled by Site-Specific Lentiviral Magnetic Targeting of SHP-2.

Hypoxia promotes vascularization by stabilization and activation of the hypoxia inducible factor 1α (HIF-1α), which constitutes a target for angiogenic gene therapy. However, gene therapy is hampered by low gene delivery efficiency and non-specific side effects. Here, we developed a gene transfer technique based on magnetic targeting of magnetic nanoparticle-lentivirus (MNP-LV) complexes allowing site-directed gene delivery to individual wounds in the dorsal skin of mice. Using this technique, we were able to control HIF-1α dependent wound healing angiogenesis in vivo via site-specific modulation of the tyrosine phosphatase activity of SHP-2. We thus uncover a novel physiological role of SHP-2 in protecting HIF-1α from proteasomal degradation via a Src kinase dependent mechanism, resulting in HIF-1α DNA-binding and transcriptional activity in vitro and in vivo. Excitingly, using targeting of MNP-LV complexes, we achieved simultaneous expression of constitutively active as well as inactive SHP-2 mutant proteins in separate wounds in vivo and hereby specifically and locally controlled HIF-1α activity as well as the angiogenic wound healing response in vivo. Therefore, magnetically targeted lentiviral induced modulation of SHP-2 activity may be an attractive approach for controlling patho-physiological conditions relying on hypoxic vessel growth at specific sites.

The proteasome regulates collagen-induced platelet aggregation via nuclear-factor-kappa-B (NFĸB) activation.

INTRODUCTION: Platelets possess critical hemostatic functions in the system of thrombosis and hemostasis, which can be affected by a multitude of external factors. Previous research has shown that platelets have the capacity to synthesize proteins de novo and more recently a multicatalytic protein complex, the proteasome, has been discovered in platelets. Due to its vital function for cellular integrity, the proteasome has become a therapeutic target for anti-proliferative drug therapies in cancer. Clinically thrombocytopenia is a frequent side-effect, but the aggregatory function of platelets also appears to be affected. Little is known however about underlying regulatory mechanisms and functional aspects of proteasome inhibition on platelets. Our study aims to investigate the role of the proteasome in regulating collagen-induced platelet aggregation and its interaction with NFkB in this context. MATERIAL AND METHODS: Using fluorescence activity assays, platelet aggregometry and immunoblotting, we investigate regulatory interactions of the proteasome and Nuclear-factor-kappa-B (NFkB) in collagen-induced platelet aggregation. RESULTS: We show that collagen induces proteasome activation in platelets and collagen-induced platelet aggregation can be reduced with proteasome inhibition by the specific inhibitor epoxomicin. This effect does not depend on Rho-kinase/ROCK activation or thromboxane release, but rather depends on NFkB activation. Inhibition of the proteasome prevented cleavage of NFκB-inhibitor protein IκBα and decreased NFκB activity after collagen stimulation. Inhibition of the NFκB-pathway in return reduced collagen-induced platelet proteasome activity and cleavage of proteasome substrates. CONCLUSIONS: This work offers novel explanations how the proteasome influences collagen-dependent platelet aggregation by involving non-genomic functions of NFkB.

Impaired endothelial shear stress induces podosome assembly via VEGF up-regulation.

Podosomes are dynamic cytoskeletal membrane structures with local adhesive and proteolytic activity. They are critically involved in angiogenesis and vascular adaptive growth. Here, we studied in HUVECs and murine small vessels whether shear stress controls podosome assembly and local proteolytic activity. Podosomes were characterized by immunohistochemistry, and their proteolytic activity was assessed as degradation imprints in fluorescent gelatin that was used as growth substrate. Compared with controls (10 dyn/cm(2)), the number of podosomes formed per time was doubled when cells were exposed to low shear stress (0.3 dyn/cm(2)) or even increased 5-fold under static conditions. This was a result of an enhanced expression of VEGF after reduction of shear stress. Consequently, enhanced podosome formation could be prevented by a VEGF receptor antagonist as well by interruption of VEGF signaling via inhibition of PI3K, Src, or p38. Increase of podosome assembly went along with significantly augmented cell motility. In vivo experiments in mouse arteries confirmed increased endothelial podosome numbers when shear stress was abolished by vessel occlusion. We conclude that shear stress, by reducing VEGF release, inhibits podosome assembly. Hence, endothelial cell-mediated matrix proteolysis and migratory activity are inhibited, thereby stabilizing the structure of the vessel wall.-Fey, T., Schubert, K. M., Schneider, H., Fein, E., Kleinert, E., Pohl, U., Dendorfer, A. Impaired endothelial shear stress induces podosome assembly via VEGF up-regulation.

AMPK Dilates Resistance Arteries via Activation of SERCA and BKCa Channels in Smooth Muscle.

The protective effects of 5'-AMP-activated protein kinase (AMPK) on the metabolic syndrome may include direct effects on resistance artery vasomotor function. However, the precise actions of AMPK on microvessels and their potential interaction are largely unknown. Thus, we set to determine the effects of AMPK activation on vascular smooth muscle tone and the underlying mechanisms. Resistance arteries isolated from hamster and mouse exhibited a pronounced endothelium-independent dilation on direct pharmacological AMPK activation by 2 structurally unrelated compounds (PT1 and A769662). The dilation was associated with a decrease of intracellular-free calcium [Ca(2+)]i in vascular smooth muscle cell. AMPK stimulation induced activation of BKCa channels as assessed by patch clamp studies in freshly isolated hamster vascular smooth muscle cell and confirmed by direct proof of membrane hyperpolarization in intact arteries. The BKCa channel blocker iberiotoxin abolished the hyperpolarization but only partially reduced the dilation and did not affect the decrease of [Ca(2+)]i. By contrast, the sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA) inhibitor thapsigargin largely reduced these effects, whereas combined inhibition of SERCA and BKCa channels virtually abolished them. AMPK stimulation significantly increased the phosphorylation of the SERCA modulator phospholamban at the regulatory T17 site. Stimulation of smooth muscle AMPK represents a new, potent vasodilator mechanism in resistance vessels. AMPK directly relaxes vascular smooth muscle cell by a decrease of [Ca(2+)]i. This is achieved by calcium sequestration via SERCA activation, as well as activation of BKCa channels. There is in part a mutual compensation of both calcium-lowering mechanisms. However, SERCA activation which involves an AMPK-dependent phosphorylation of phospholamban is the predominant mechanism in resistance vessels.

Knockout of mitochondrial thioredoxin reductase stabilizes prolyl hydroxylase 2 and inhibits tumor growth and tumor-derived angiogenesis.

AIMS: Mitochondrial thioredoxin reductase (Txnrd2) is a central player in the control of mitochondrial hydrogen peroxide (H2O2) abundance by serving as a direct electron donor to the thioredoxin-peroxiredoxin axis. In this study, we investigated the impact of targeted disruption of Txnrd2 on tumor growth. RESULTS: Tumor cells with a Txnrd2 deficiency failed to activate hypoxia-inducible factor-1α (Hif-1α) signaling; it rather caused PHD2 accumulation, Hif-1α degradation and decreased vascular endothelial growth factor (VEGF) levels, ultimately leading to reduced tumor growth and tumor vascularization. Increased c-Jun NH2-terminal Kinase (JNK) activation proved to be the molecular link between the loss of Txnrd2, an altered mitochondrial redox balance with compensatory upregulation of glutaredoxin-2, and elevated PHD2 expression. INNOVATION: Our data provide compelling evidence for a yet-unrecognized mitochondrial Txnrd-driven, regulatory mechanism that ultimately prevents cellular Hif-1α accumulation. In addition, simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems was used as an efficient therapeutic approach in hindering tumor growth. CONCLUSION: This work demonstrates an unexpected regulatory link between mitochondrial Txnrd and the JNK-PHD2-Hif-1α axis, which highlights how the loss of Txnrd2 and the resulting altered mitochondrial redox balance impairs tumor growth as well as tumor-related angiogenesis. Furthermore, it opens a new avenue for a therapeutic approach to hinder tumor growth by the simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems.

Angiopoietin 2 mediates microvascular and hemodynamic alterations in sepsis.

Septic shock is characterized by increased vascular permeability and hypotension despite increased cardiac output. Numerous vasoactive cytokines are upregulated during sepsis, including angiopoietin 2 (ANG2), which increases vascular permeability. Here we report that mice engineered to inducibly overexpress ANG2 in the endothelium developed sepsis-like hemodynamic alterations, including systemic hypotension, increased cardiac output, and dilatory cardiomyopathy. Conversely, mice with cardiomyocyte-restricted ANG2 overexpression failed to develop hemodynamic alterations. Interestingly, the hemodynamic alterations associated with endothelial-specific overexpression of ANG2 and the loss of capillary-associated pericytes were reversed by intravenous injections of adeno-associated viruses (AAVs) transducing cDNA for angiopoietin 1, a TIE2 ligand that antagonizes ANG2, or AAVs encoding PDGFB, a chemoattractant for pericytes. To confirm the role of ANG2 in sepsis, we i.p. injected LPS into C57BL/6J mice, which rapidly developed hypotension, acute pericyte loss, and increased vascular permeability. Importantly, ANG2 antibody treatment attenuated LPS-induced hemodynamic alterations and reduced the mortality rate at 36 hours from 95% to 61%. These data indicate that ANG2-mediated microvascular disintegration contributes to septic shock and that inhibition of the ANG2/TIE2 interaction during sepsis is a potential therapeutic target.

Combined deficiency in glutathione peroxidase 4 and vitamin E causes multiorgan thrombus formation and early death in mice.

RATIONALE: Growing evidence indicates that oxidative stress contributes markedly to endothelial dysfunction. The selenoenzyme glutathione peroxidase 4 (Gpx4) is an intracellular antioxidant enzyme important for the protection of membranes by its unique activity to reduce complex hydroperoxides in membrane bilayers and lipoprotein particles. Yet a role of Gpx4 in endothelial cell function has remained enigmatic. OBJECTIVE: To investigate the role of Gpx4 ablation and subsequent lipid peroxidation in the vascular compartment in vivo. METHODS AND RESULTS: Endothelium-specific deletion of Gpx4 had no obvious impact on normal vascular homeostasis, nor did it impair tumor-derived angiogenesis in mice maintained on a normal diet. In stark contrast, aortic explants from endothelium-specific Gpx4 knockout mice showed a markedly reduced number of endothelial branches in sprouting assays. To shed light onto this apparent discrepancy between the in vivo and ex vivo results, we depleted mice of a second antioxidant, vitamin E, which is normally absent under ex vivo conditions. Therefore, mice were fed a vitamin E-depleted diet for 6 weeks before endothelial deletion of Gpx4 was induced by 4-hydroxytamoxifen. Surprisingly, ≈80% of the knockout mice died. Histopathological analysis revealed detachment of endothelial cells from the basement membrane and endothelial cell death in multiple organs, which triggered thrombus formation. Thromboembolic events were the likely cause of various clinical pathologies, including heart failure, renal and splenic microinfarctions, and paraplegia. CONCLUSIONS: Here, we show for the first time that in the absence of Gpx4, sufficient vitamin E supplementation is crucial for endothelial viability.

Hydrogen sulfide-releasing aspirin derivative ACS14 exerts strong antithrombotic effects in vitro and in vivo.

OBJECTIVE: Hydrogen sulfide (H(2)S)-releasing NSAIDs exert potent anti-inflammatory effects beyond classical cyclooxygenase inhibition. Here, we compared the platelet inhibitory effects of the H(2)S-releasing aspirin derivative ACS14 with its mother compound aspirin to analyze additional effects on platelets. METHODS AND RESULTS: In platelets of mice fed with ACS14 for 6 days (50 mg/kg per day), not only arachidonic acid-induced platelet aggregation but also ADP-dependent aggregation was decreased, an effect that was not observed with an equimolar dose of aspirin (23 mg/kg per day). ACS14 led to a significantly longer arterial occlusion time after light-dye-induced endothelial injury as well as decreased thrombus formation after ferric chloride-induced injury in the carotid artery. Bleeding time was not prolonged compared with animals treated with equimolar doses of aspirin. In vitro, in human whole blood, ACS14 (25-500 µmol/L) inhibited arachidonic acid-induced platelet aggregation, but compared with aspirin additionally reduced thrombin receptor-activating peptide-, ADP-, and collagen-dependent aggregation. In washed human platelets, ACS14 (500 µmol/L) attenuated αIIbß3 integrin activation and fibrinogen binding and increased intracellular cAMP levels and cAMP-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation. CONCLUSIONS: The H(2)S-releasing aspirin derivative ACS14 exerts strong antiaggregatory effects by impairing the activation of the fibrinogen receptor by mechanisms involving increased intracellular cyclic nucleotides. These additional antithrombotic properties result in a more efficient inhibition of thrombus formation in vivo as achieved with aspirin alone.

ARNO regulates VEGF-dependent tissue responses by stabilizing endothelial VEGFR-2 surface expression.

AIMS: The vascular endothelial growth factor (VEGF) stimulates angiogenesis by induction of vessel permeability, proliferation, and migration of endothelial cells, an important process in ischaemic diseases. ADP-ribosylation factor (ARF) nucleotide-binding site opener (ARNO) (cytohesin-2) is a guanine exchange factor important for cellular signalling through ARF GTPases. However, a role for ARNO in VEGF-dependent endothelial processes has so far not been documented. Therefore, we investigated whether ARNO has a role in VEGF-dependent activation of endothelial cells and thus vessel permeability. METHODS AND RESULTS: ARNO expression was observed in endothelial cells in vitro by RT-PCR, western blotting, and immunofluorescence as well as ex vivo by immunohistochemical staining of mouse aorta. Treatment with the cytohesin inhibitor SecinH3 or with an ARNO siRNA prevented VEGF-dependent Akt activation, assessed by detection of phosphorylated Akt, and proliferation of endothelial cells in vitro, measured by methylthiazoletetrazolium (MTT) reduction. In addition, ARNO suppression reduced VEGF-induced permeability in vessels of the mouse (C57BL/6) cremaster muscle in vivo, as measured by extravasation of fluorescein isothiocyanate (FITC)-dextran. Moreover, ARNO knock-down accelerated ligand-induced reduction in vascular endothelial growth factor receptor-2 (VEGFR-2) surface expression, internalization, and degradation, as assessed by flow cytometry and western blotting, respectively. CONCLUSION: Our findings indicate an important and novel role for endothelial ARNO in VEGF-dependent initiation of angiogenesis by regulation of VEGFR-2 internalization in endothelial cells, resulting in the activation of the Akt pathway, vessel permeability, and ultimately endothelial proliferation. Thus, ARNO may be a new essential player in endothelial signalling and angiogenesis.

Gap junctional communication controls the overall endothelial calcium response to vasoactive agonists.

AIMS: A cytosolic calcium (Ca(2+)(i)) increase is an important activation signal for the endothelium. We investigated whether interendothelial spreading of the Ca(2+) signal via gap junctions (GJs) plays a role for the overall Ca(2+)(i) increase in response to vasoactive agonists. METHODS AND RESULTS: In human umbilical vein endothelial cells (HUVECs), a Ca(2+)(i) increase (Fura2) in response to histamine or ATP occurred initially only in about 30% of the cells (initially responding cells) reflecting the cell fraction expressing H(1) or purinergic receptors (FACS/immunohistochemistry). In the remaining adjacent cells, Ca(2+)(i) increases occurred only after a delay of up to 5 s. Blockade of GJ communication (meclofenamic acid and heptanol, or H(2)O(2); verified by dye injection) did not affect responses in the initially responding cells but abolished the delayed Ca(2+)(i) response of the remaining adjacent cells. The resulting reduction in the global endothelial Ca(2+)(i) response significantly reduced the nitric oxide synthesis (assessed as cGMP levels). Similar Ca(2+)(i) results were obtained in the endothelium of freshly isolated mouse (C57BL/6) aortas stimulated with ATP. The receptor-independent Ca(2+)(i) response to ionomycin occurred simultaneously in all cells, regardless of GJ inhibition. In separate experiments, inhibition of the IP(3) receptor (xestospongin-C; 40, µmol/L) but not of the ryanodine receptor (ryanodine, 250 µmol/L) reduced the spread of the Ca(2+)(i) signal into adjacent cells over longer distances. CONCLUSION: The global Ca(2+)(i) response of the endothelium to agonists is determined decisively by the functionality of GJs, thus establishing a new role for GJs in controlling endothelial activity and vasomotor function.

The carboxyl tail of Cx43 augments p38 mediated cell migration in a gap junction-independent manner.

The expression of connexin 43 (Cx43) has been shown to correlate with an enhanced migration of several cell types such as glioma or neural crest cells, but the mechanism remains unclear. We studied whether Cx43 also affects migration in non-neural cells and whether or not this is related to gap junction formation. Therefore, we analysed the migratory activity of HeLa cells under conditions of controlled connexin (Cx) expression. The expression of Cx43 enhanced their migration significantly as compared to Cx deficient wild-type cells. Expression of only the carboxyl tail of Cx43 (Cx43CT, AA 257-382) without channel forming capacity enhanced migration similarly as the full length protein. In contrast, the expression of the N-terminal part of Cx43 (Cx43NT, AA 1-257), which partially retained the gap junction channel function of Cx43, did not increase migration. The enhanced cell migration of HeLa cells expressing either full length Cx43 or the Cx43CT was associated with an increased activation of the p38 MAP kinase. The additional incubation with a specific inhibitor of p38 activation diminished the migration of HeLa-Cx43 cells to levels of control transfected cells. As a proof of concept, we studied whether Cx43 also modulates the migration of endothelial progenitor cells (EPC) which play an important role in angiogenesis. In these cells, which expressed Cx43 as the only connexin, the downregulation of Cx43 by siRNA resulted in a significantly decreased migration. These results demonstrate that expression of Cx43 augments migration via modulation of p38 MAP kinase activity. The carboxyl tail of Cx43 plays an essential role in this signalling pathway which is independent of gap junction function.

Suppression of DNA-PKcs enhances FGF-2 dependent human endothelial cell proliferation via negative regulation of Akt.

Angiogenesis initiation is crucially dependent on endothelial proliferation and can be stimulated by the fibroblast growth factor 2 (FGF-2). The DNA dependent protein kinase (DNA-PK), long known for its importance in repairing DNA double strand breaks, belongs to the phosphatidylinositol-3 kinase (PI3-K) super family and has recently been identified as one of the enzymes phosphorylating and activating Akt. Due to its similarity with PI3-K, we hypothesized that DNA-PK may have similar effects on endothelial angiogenic processes and signalling. We used primary endothelial cells (HUVEC and PAEC) and human microvascular endothelial cells (HMEC) to study the role of DNA-PK in endothelial proliferation and signalling. DNA-PKcs suppression with the compound NU7026 or with siRNA induced basal endothelial cell proliferation as well as enhanced FGF-2 dependent proliferation. This was associated with an increase in phosphorylated Akt. Tube formation was not affected by DNA-PKcs clearly showing that the role of DNA-PK in endothelial processes differs from that of PI3-K. Our findings indicate DNA-PK as an important enzyme maintaining the quiescent endothelial phenotype by actively inhibiting Akt thus restraining endothelial cell proliferation preventing excessive growth.

Role of sphingosine-1-phosphate phosphohydrolase 1 in the regulation of resistance artery tone.

Sphingosine-1-phosphate (S1P), which mediates pleiotropic actions within the vascular system, is a prominent regulator of microvascular tone. By virtue of its S1P-degrading function, we hypothesized that S1P-phosphohydrolase 1 (SPP1) is an important regulator of tone in resistance arteries. Hamster gracilis muscle resistance arteries express mRNA encoding SPP1. Overexpression of SPP1 (via transfection of a SPP1(wt)) reduced resting tone, Ca2+ sensitivity, and myogenic vasoconstriction, whereas reduced SPP1 expression (antisense oligonucleotides) yielded the opposite effects. Expression of a phosphatase-dead mutant of SPP1 (SPP1(H208A)) had no effect on any parameter tested, suggesting that catalytic activity of SPP1 is critical. The enhanced myogenic tone that follows overexpression of S1P-generating enzyme sphingosine kinase 1 (Sk1(wt)) was functionally antagonized by coexpression with SPP1(wt) but not SPP1(H208A). SPP1 modulated vasoconstriction in response to 1 to 100 nmol/L exogenous S1P, a concentration range that was characterized as S1P2-dependent, based on the effect of S1P(2) inhibition by antisense oligonucleotides and 1 mumol/L JTE013. Inhibition of the cystic fibrosis transmembrane regulator (CFTR) (1) restored S1P responses that were attenuated by SPP1(wt) overexpression; (2) enhanced myogenic vasoconstriction; but (3) had no effect on noradrenaline responses. We conclude that SPP1 is an endogenous regulator of resistance artery tone that functionally antagonizes the vascular effects of both Sk1(wt) and S1P2 receptor activation. SPP1 accesses extracellular S1P pools in a manner dependent on a functional CFTR transport protein. Our study assigns important roles to both SPP1 and CFTR in the physiological regulation of vascular tone, which influences both tissue perfusion and systemic blood pressure.

Inhibition of the tyrosine phosphatase SHP-2 suppresses angiogenesis in vitro and in vivo.

Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new vessel formation. We investigated the role of SHP-2 in endothelial cell survival and angiogenesis in vitro as well as in vivo. SHP-2 function in cultured human umbilical vein and human dermal microvascular endothelial cells was inhibited by either silencing the protein expression with antisense-oligodesoxynucleotides or treatment with a pharmacological inhibitor (PtpI IV). SHP-2 inhibition impaired capillary-like structure formation (p < 0.01; n = 8) in vitro as well as new vessel growth ex vivo(p < 0.05; n = 10) and in vivo in the chicken chorioallantoic membrane (p < 0.01, n = 4). Additionally, SHP-2 knock-down abrogated fibroblast growth factor 2 (FGF-2)-dependent endothelial proliferation measured by MTT reduction (p < 0.01; n = 12). The inhibitory effect of SHP-2 knock-down on vessel growth was mediated by increased endothelial apoptosis (annexin V staining, p < 0.05, n = 9), which was associated with reduced FGF-2-induced phosphorylation of phosphatidylinositol 3-kinase (PI3-K), Akt and extracellular regulated kinase 1/2 (ERK1/2) and involved diminished ERK1/2 phosphorylation after PI3-K inhibition (n = 3). These results suggest that SHP-2 regulates endothelial cell survival through PI3-K-Akt and mitogen-activated protein kinase pathways thereby strongly affecting new vessel formation. Thus, SHP-2 exhibits a pivotal role in angiogenesis and may represent an interesting target for therapeutic approaches controlling vessel growth.

Endogenous and exogenous NO attenuates conduction of vasoconstrictions along arterioles in the microcirculation.

Vascular coordination in the microcirculation depends on gap junctional intercellular communication (GJIC), which is reflected by the conduction of locally initiated vasomotor responses. However, little is known about the regulation of GJIC in vivo. We hypothesized that endothelial NO regulates GJIC and therefore studied whether conduction of constrictions and dilations along the vessel wall is modulated by modifying the level of microcirculatory NO. Arterioles were focally stimulated using high K(+) or acetylcholine in the cremaster muscle in situ, and diameter changes were assessed at the local and remote upstream sites by intravital microscopy. Local stimulation with K(+) initiated a constriction that conducted along the arteriole with diminishing amplitude (length constant lambda: 371 +/- 42 mum). After N(omega)-nitro-l-arginine (l-NNA), lambda increased to 507 +/- 30 mum, indicating that GJIC is attenuated by endogenous NO. Exogenous NO, but not adenosine, reduced lambda after l-NNA in a reversible, concentration-dependent, and mainly cGMP-dependent manner as assessed by inhibition of soluble guanylate cyclase. In endothelial NO synthase-deficient mice, lambda was 530 +/- 80 mum and thus similar to that in wild-type mice after l-NNA. Exogenous NO likewise reduced lambda in these mice. The effects of NO were comparable to those of wild-type animals in Cx40-deficient mice, which excludes Cx40 as a specific target of NO. In contrast to constrictions, the amplitude of conducted dilations on acetylcholine did not diminish up to 1,300 mum and were not altered by l-NNA or exogenous NO. We conclude that endogenously released NO attenuates the conduction of vasoconstrictions most likely due to a modulation of gap junctional conductivity. We suggest that this effect is specific for smooth muscle cells, which probably transmit constricting signals, and involves connexins other than Cx40. This mechanism may support the dilatory potency of NO by preventing the conduction of remote vasoconstrictions into areas with basal or activated NO release.

The tyrosine phosphatase, SHP-1, is a negative regulator of endothelial superoxide formation.

OBJECTIVES: We investigated the role of SH2-domain containing phosphatase-1 (SHP-1) in endothelial reduced nicotinamide adenine dinucleotide (phosphate) (NAD[P]H)-oxidase-dependent oxidant production. BACKGROUND: Superoxide (O2*-) generation by endothelial NAD(P)H-oxidase promotes endothelial dysfunction and atherosclerosis. Signaling pathways that regulate NAD(P)H-oxidase activity are, however, poorly understood. METHODS: SH2-domain containing phosphatase-1 was inhibited using site-directed magnetofection of antisense oligodesoxynucleotides (AS-ODN) or short interfering ribonucleic acid (siRNA) in vitro in human umbilical vein endothelial cells (HUVEC) and in isolated hamster arteries; O2*- was measured by cytochrome c reduction in vitro. Activities of NAD(P)H-oxidase activity, phosphatidyl-inositol-3-kinase (PI3K), and SHP-1 were assessed by specific assays; Rac1 activation was assessed by a pull-down assay. RESULTS: Basal endothelial O2*- release was enhanced after inhibition of endothelial SHP-1 (p < 0.01), which could be prevented by specific inhibition of NAD(P)H-oxidase (p < 0.01); SHP-1 activity was high under basal conditions, further increased by vascular endothelial growth factor (10 ng/ml, p < 0.05), and abolished by SHP-1 AS-ODN treatment (p < 0.01), which also increased NAD(P)H-oxidase activity 3.3-fold (p < 0.01). Vascular endothelial growth factor also induced O2*- release (p < 0.01), which was even more enhanced when SHP-1 was knocked down (p < 0.05). The effect of SHP-1 was mediated by inhibition of PI3K/Rac1-dependent NAD(P)H-oxidase activation (p < 0.01); SHP-1 AS-ODN augmented tyrosine phosphorylation of the p85 regulatory subunit of PI3K (p < 0.05) and Rac1 activation. The latter was prevented by wortmannin, a blocker of PI3K. CONCLUSIONS: In HUVEC, SHP-1 counteracts basal and stimulated NAD(P)H-oxidase activity by negative regulation of PI3K-dependent Rac1 activation; SHP-1 thus seems to be an important part of endothelial antioxidative defense controlling the activity of the O2(*-)-producing NAD(P)H-oxidase.