Connexins: Key Players in the Control of Vascular Plasticity and Function.

Of the 21 members of the connexin family, 4 (Cx37, Cx40, Cx43, and Cx45) are expressed in the endothelium and/or smooth muscle of intact blood vessels to a variable and dynamically regulated degree. Full-length connexins oligomerize and form channel structures connecting the cytosol of adjacent cells (gap junctions) or the cytosol with the extracellular space (hemichannels). The different connexins vary mainly with regard to length and sequence of their cytosolic COOH-terminal tails. These COOH-terminal parts, which in the case of Cx43 are also translated as independent short isoforms, are involved in various cellular signaling cascades and regulate cell functions. This review focuses on channel-dependent and -independent effects of connexins in vascular cells. Channels play an essential role in coordinating and synchronizing endothelial and smooth muscle activity and in their interplay, in the control of vasomotor actions of blood vessels including endothelial cell reactivity to agonist stimulation, nitric oxide-dependent dilation, and endothelial-derived hyperpolarizing factor-type responses. Further channel-dependent and -independent roles of connexins in blood vessel function range from basic processes of vascular remodeling and angiogenesis to vascular permeability and interactions with leukocytes with the vessel wall. Together, these connexin functions constitute an often underestimated basis for the enormous plasticity of vascular morphology and function enabling the required dynamic adaptation of the vascular system to varying tissue demands.

The mitochondrial thioredoxin reductase system (TrxR2) in vascular endothelium controls peroxynitrite levels and tissue integrity.

The mitochondrial thioredoxin/peroxiredoxin system encompasses NADPH, thioredoxin reductase 2 (TrxR2), thioredoxin 2, and peroxiredoxins 3 and 5 (Prx3 and Prx5) and is crucial to regulate cell redox homeostasis via the efficient catabolism of peroxides (TrxR2 and Trxrd2 refer to the mitochondrial thioredoxin reductase protein and gene, respectively). Here, we report that endothelial TrxR2 controls both the steady-state concentration of peroxynitrite, the product of the reaction of superoxide radical and nitric oxide, and the integrity of the vascular system. Mice with endothelial deletion of the Trxrd2 gene develop increased vascular stiffness and hypertrophy of the vascular wall. Furthermore, they suffer from renal abnormalities, including thickening of the Bowman's capsule, glomerulosclerosis, and functional alterations. Mechanistically, we show that loss of Trxrd2 results in enhanced peroxynitrite steady-state levels in both vascular endothelial cells and vessels by using a highly sensitive redox probe, fluorescein-boronate. High steady-state peroxynitrite levels were further found to coincide with elevated protein tyrosine nitration in renal tissue and a substantial change of the redox state of Prx3 toward the oxidized protein, even though glutaredoxin 2 (Grx2) expression increased in parallel. Additional studies using a mitochondria-specific fluorescence probe (MitoPY1) in vessels revealed that enhanced peroxynitrite levels are indeed generated in mitochondria. Treatment with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin [Mn(III)TMPyP], a peroxynitrite-decomposition catalyst, blunted intravascular formation of peroxynitrite. Our data provide compelling evidence for a yet-unrecognized role of TrxR2 in balancing the nitric oxide/peroxynitrite ratio in endothelial cells in vivo and thus establish a link between enhanced mitochondrial peroxynitrite and disruption of vascular integrity.

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.

Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2.

The gap junction protein connexin 43 (Cx43) is associated with increased cell migration and to related changes of the actin cytoskeleton, which is mediated via its C-terminal cytoplasmic tail and is independent of its channel function. Cx43 has been shown to possess an angiogenic potential, however, the role of Cx43 in endothelial cell migration has not yet been investigated. Here, we found that the knock-down of Cx43 by siRNA in human microvascular endothelial cells (HMEC) reduces migration, as assessed by a wound assay in vitro and impaired aortic vessel sprouting ex vivo. Immunoprecipitation of Cx43 revealed an interaction with the tyrosine phosphatase SHP-2, which enhanced its phosphatase activity, as observed in Cx43 expressing HeLa cells compared to cells treated with an empty vector. Interestingly, the expression of a dominant negative substrate trapping mutant SHP-2 (CS) in HMEC, via lentiviral transduction, also impaired endothelial migration to a similar extent as Cx43 siRNA compared to SHP-2 WT. Moreover, the reduction in endothelial migration upon Cx43 siRNA could not be rescued by the introduction of a constitutively active SHP-2 construct (EA). Our data demonstrate that Cx43 and SHP-2 mediate endothelial cell migration, revealing a novel interaction between Cx43 and SHP-2, which is essential for this process.

Estimating hemodynamic shear stress in murine peripheral collateral arteries by two-photon line scanning.

Changes in wall shear stress of blood vessels are assumed to be an important component of many physiological and pathophysiological processes. However, due to technical limitations experimental in vivo data are rarely available. Here, we investigated two-photon excitation fluorescence microscopy as an option to measure vessel diameter as well as blood flow velocities in a murine hindlimb model of arteriogenesis (collateral artery growth). Using line scanning at high frequencies, we measured the movement of blood cells along the vessel axis. We found that peak systolic blood flow velocity averaged 9 mm/s and vessel diameter 42 µm in resting collaterals. Induction of arteriogenesis by femoral artery ligation resulted in a significant increase in centerline peak systolic velocity after 1 day with an average of 51 mm/s, whereas the averaged luminal diameter of collaterals (52 µm) changed much less. Thereof calculations revealed a significant fourfold increase in hemodynamic wall shear rate. Our results indicate that two-photon line scanning is a suitable tool to estimate wall shear stress e.g., in experimental animal models, such as of arteriogenesis, which may not only help to understand the relevance of mechanical forces in vivo, but also to adjust wall shear stress in ex vivo investigations on isolated vessels as well as cell culture experiments.

The AMP-Related Kinase (AMPK) Induces Ca2+-Independent Dilation of Resistance Arteries by Interfering With Actin Filament Formation.

RATIONALE: Decreasing Ca2+ sensitivity of vascular smooth muscle (VSM) allows for vasodilation without lowering of cytosolic Ca2+. This may be particularly important in states requiring maintained dilation, such as hypoxia. AMP-related kinase (AMPK) is an important cellular energy sensor in VSM. Regulation of Ca2+ sensitivity usually is attributed to myosin light chain phosphatase activity, but findings in non-VSM identified changes in the actin cytoskeleton. The potential role of AMPK in this setting is widely unknown. OBJECTIVE: To assess the influence of AMPK on the actin cytoskeleton in VSM of resistance arteries with regard to potential Ca2+ desensitization of VSM contractile apparatus. METHODS AND RESULTS: AMPK induced a slowly developing dilation at unchanged cytosolic Ca2+ levels in potassium chloride-constricted intact arteries isolated from mouse mesenteric tissue. This dilation was not associated with changes in phosphorylation of myosin light chain or of myosin light chain phosphatase regulatory subunit. Using ultracentrifugation and confocal microscopy, we found that AMPK induced depolymerization of F-actin (filamentous actin). Imaging of arteries from LifeAct mice showed F-actin rarefaction in the midcellular portion of VSM. Immunoblotting revealed that this was associated with activation of the actin severing factor cofilin. Coimmunoprecipitation experiments indicated that AMPK leads to the liberation of cofilin from 14-3-3 protein. CONCLUSIONS: AMPK induces actin depolymerization, which reduces vascular tone and the response to vasoconstrictors. Our findings demonstrate a new role of AMPK in the control of actin cytoskeletal dynamics, potentially allowing for long-term dilation of microvessels without substantial changes in cytosolic Ca2+.

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.

NO Augments Endothelial Reactivity by Reducing Myoendothelial Calcium Signal Spreading: A Novel Role for Cx37 (Connexin 37) and the Protein Tyrosine Phosphatase SHP-2.

OBJECTIVE: Because of its strategic position between endothelial and smooth muscle cells in microvessels, Cx37 (Connexin 37) plays an important role in myoendothelial gap junctional intercellular communication. We have shown before that NO inhibits gap junctional intercellular communication through gap junctions containing Cx37. However, the underlying mechanism is not yet identified. APPROACH AND RESULTS: Using channel-forming Cx37 mutants exhibiting partial deletions or amino acid exchanges in their C-terminal loops, we now show that the phosphorylation state of a tyrosine residue at position 332 (Y332) in the C-terminus of Cx37 controls the gap junction-dependent spread of calcium signals. Mass spectra revealed that NO protects Cx37 from dephosphorylation at Y332 by inhibition of the protein tyrosine phosphatase SHP-2. Functionally, the inhibition of gap junctional intercellular communication by NO decreased the spread of the calcium signal (induced by mechanical stimulation of individual endothelial cells) from endothelial to smooth muscle cells in intact vessels, while, at the same time, augmenting the calcium signal spreading within the endothelium. Consequently, preincubation of small resistance arteries with exogenous NO enhanced the endothelium-dependent dilator response to acetylcholine in spite of a pharmacological blockade of NO-dependent cGMP formation by the soluable guanylyl cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). CONCLUSIONS: Our results identify a novel mechanism by which NO can increase the efficacy of calcium, rising vasoactive agonists in the microvascular endothelium.

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.

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.

Endothelial alpha-parvin controls integrity of developing vasculature and is required for maintenance of cell-cell junctions.

RATIONALE: Angiogenesis and vessel integrity depend on the adhesion of endothelial cells (ECs) to the extracellular matrix and to adjacent ECs. The focal adhesion protein α-parvin (α-pv) is essential for vascular development. However, the role of α-pv in ECs in vivo is not known. OBJECTIVE: To determine the function of α-pv in ECs during vascular development in vivo and the underlying mechanisms. METHODS AND RESULTS: We deleted the α-pv gene specifically in ECs of mice to study its role in angiogenesis and vascular development. Here, we show that endothelial-specific deletion of α-pv in mice results in late embryonic lethality associated with hemorrhages and reduced vascular density. Postnatal-induced EC-specific deletion of α-pv leads to retinal hypovascularization because of reduced vessel sprouting and excessive vessel regression. In the absence of α-pv, blood vessels display impaired VE-cadherin junction morphology. In vitro, α-pv-deficient ECs show reduced stable adherens junctions, decreased monolayer formation, and impaired motility, associated with reduced formation of integrin-mediated cell-extracellular matrix adhesion structures and an altered actin cytoskeleton. CONCLUSIONS: Endothelial α-pv is essential for vessel sprouting and for vessel stability.

Endothelial Dysfunction, and A Prothrombotic, Proinflammatory Phenotype Is Caused by Loss of Mitochondrial Thioredoxin Reductase in Endothelium.

OBJECTIVE: Although the investigation on the importance of mitochondria-derived reactive oxygen species (ROS) in endothelial function has been gaining momentum, little is known on the precise role of the individual components involved in the maintenance of a delicate ROS balance. Here we studied the impact of an ongoing dysregulated redox homeostasis by examining the effects of endothelial cell-specific deletion of murine thioredoxin reductase 2 (Txnrd2), a key enzyme of mitochondrial redox control. APPROACH AND RESULTS: We analyzed the impact of an inducible, endothelial cell-specific deletion of Txnrd2 on vascular remodeling in the adult mouse after femoral artery ligation. Laser Doppler analysis and histology revealed impaired angiogenesis and arteriogenesis. In addition, endothelial loss of Txnrd2 resulted in a prothrombotic, proinflammatory vascular phenotype, manifested as intravascular cellular deposits, as well as microthrombi. This phenotype was confirmed by an increased leukocyte response toward interleukin-1 in the mouse cremaster model. In vitro, we could confirm the attenuated angiogenesis measured in vivo, which was accompanied by increased ROS and an impaired mitochondrial membrane potential. Ex vivo analysis of femoral arteries revealed reduced flow-dependent vasodilation in endothelial cell Txnrd2-deficient mice. This endothelial dysfunction could be, at least partly, ascribed to inadequate nitric oxide signaling. CONCLUSIONS: We conclude that the maintenance of mitochondrial ROS via Txnrd2 in endothelial cells is necessary for an intact vascular homeostasis and remodeling and that Txnrd2 plays a vitally important role in balancing mitochondrial ROS production in the endothelium.

Cx43 increases serum induced filopodia formation via activation of p21-activated protein kinase 1.

In a previous study we could show that connexin 43 (Cx43) expression increased the migration of cells in a channel-independent manner involving the MAPK p38. We analyzed here the mechanism by which Cx43 enhanced p38 activation and migration related changes of the actin cytoskeleton. HeLa cells were used as a model system for the controlled expression of Cx43 and truncated Cx43 proteins. The expression of Cx43 altered the actin cytoskeleton organization in response to serum stimulation. Cx43 expressing HeLa cells had significantly more filopodial protrusions per cell than empty-vector transfected control cells. The expression of the channel incompetent carboxyl tail of Cx43 was sufficient to enhance the filopodia formation whereas the N-terminal, channel-building part, had no such effect. The enhanced filopodia formation was p38 dependent since the p38 blocker SB203580 significantly diminished it. Immunoprecipitation revealed an interaction of the upstream regulator of p38, p21-activated protein kinase 1 (PAK1), with Cx43 resulting in an enhanced phosphorylation of PAK1. Moreover, p38 activation, filopodia formation and cell migration were significantly reduced by blocking the PAK1 activity with its pharmacological inhibitor, IPA-3. The p38 target Hsp27, which favors the actin polymerization in its phosphorylated form, was significantly more phosphorylated characterizing it as a potential candidate molecule to enhance the serum-induced actin polymerization in Cx43 expressing cells. Our results provide a novel mechanism by which Cx43 can modify actin cytoskeletal dynamics and may thereby enhance cell migration.

Performance of bioactive PMMA-based bone cement under load-bearing conditions: an in vivo evaluation and FE simulation.

In the past, bioactive bone cement was investigated in order to improve the durability of cemented arthroplasties by strengthening the bone-cement interface. As direct bone-cement bonding may theoretically lead to higher stresses within the cement, the question arises, whether polymethylmethacrylate features suitable mechanical properties to withstand altered stress conditions? To answer this question, in vivo experiments and finite element simulations were conducted. Twelve rabbits were divided into two groups examining either bioactive polymethylmethacrylate-based cement with unchanged mechanical properties or commercially available polymethylmethacrylate cement. The cements were tested under load-bearing conditions over a period of 7 months, using a spacer prosthesis cemented into the femur. For the finite element analyses, boundary conditions of the rabbit femur were simulated and analyses were performed with respect to different loading scenarios. Calculations of equivalent stress distributions within the cements were applied, with a completely bonded cement surface for the bioactive cement and with a continuously interfering fibrous tissue layer for the reference cement. The bioactive cement revealed good in vivo bioactivity. In the bioactive cement group two failures (33 %), with complete break-out of the prosthesis occurred, while none in the reference group. Finite element analyses of simulated bioactive cement fixation showed an increase in maximal equivalent stress by 49.2 to 109.4 % compared to the simulation of reference cement. The two failures as well as an increase in calculated equivalent stress highlight the importance of fatigue properties of polymethylmethacrylate in general and especially when developing bioactive cements designated for load-bearing conditions.

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.

NO, via its target Cx37, modulates calcium signal propagation selectively at myoendothelial gap junctions.

BACKGROUND: Gap junctional calcium signal propagation (transfer of calcium or a calcium releasing messenger via gap junctions) between vascular cells has been shown to be involved in the control of vascular tone. We have shown before that nitric oxide (NO) inhibits gap junctional communication in HeLa cells exclusively expressing connexin 37 (HeLa-Cx37) but not in HeLa-Cx40 or HeLa-Cx43. Here we studied the effect of NO on the gap junctional calcium signal propagation in endothelial cells which, in addition to Cx37, also express Cx40 and Cx43. Furthermore, we analyzed the impact of NO on intermuscle and on myoendothelial gap junction-dependent calcium signal propagation. Since specific effects of NO at one of these three junctional areas (interendothelial/ myoendothelial/ intermuscle) may depend on a differential membrane localization of the connexins, we also studied the distribution of the vascular connexins in small resistance arteries. RESULTS: In endothelial (HUVEC) or smooth muscle cells (HUVSMC) alone, NO did not affect gap junctional Ca2+ signal propagation as assessed by analyzing the spread of Ca2+ signals after mechanical stimulation of a single cell. In contrast, at myoendothelial junctions, it decreased Ca2+ signal propagation in both directions by about 60% (co-cultures of HUVEC and HUVSMC). This resulted in a longer maintenance of calcium elevation at the endothelial side and a faster calcium signal propagation at the smooth muscle side, respectively. Immunohistochemical stainings (confocal and two-photon-microscopy) of cells in co-cultures or of small arteries revealed that Cx37 expression was relatively higher in endothelial cells adjoining smooth muscle (culture) or in potential areas of myoendothelial junctions (arteries). Accordingly, Cx37 - in contrast to Cx40 - was not only expressed on the endothelial surface of small arteries but also in deeper layers (corresponding to the internal elastic lamina IEL). Holes of the IEL where myoendothelial contacts can only occur, stained significantly more frequently for Cx37 and Cx43 than for Cx40 (endothelium) or Cx45 (smooth muscle). CONCLUSION: NO modulates the calcium signal propagation specifically between endothelial and smooth muscle cells. The effect is due to an augmented distribution of Cx37 towards myoendothelial contact areas and potentially counteracts endothelial Ca2+ signal loss from endothelial to smooth muscle cells. This targeted effect of NO may optimize calcium dependent endothelial vasomotor function.