Correlative intravital imaging of cGMP signals and vasodilation in mice

Cyclic guanosine monophosphate (cGMP) is an important signaling molecule and drug target in the cardiovascular system. It is well known that stimulation of the vascular nitric oxide (NO)-cGMP pathway results in vasodilation. However, the spatiotemporal dynamics of cGMP signals themselves and the cGMP concentrations within specific cardiovascular cell types in health, disease, and during pharmacotherapy with cGMP-elevating drugs are largely unknown. To facilitate the analysis of cGMP signaling in vivo, we have generated transgenic mice that express fluorescence resonance energy transfer (FRET)-based cGMP sensor proteins. Here, we describe two models of intravital FRET/cGMP imaging in the vasculature of cGMP sensor mice: (1) epifluorescence-based ratio imaging in resistance-type vessels of the cremaster muscle and (2) ratio imaging by multiphoton microscopy within the walls of subcutaneous blood vessels accessed through a dorsal skinfold chamber. Both methods allow simultaneous monitoring of NO-induced cGMP transients and vasodilation in living mice. Detailed protocols of all steps necessary to perform and evaluate intravital imaging experiments of the vasculature of anesthetized mice including surgery, imaging, and data evaluation are provided. An image segmentation approach is described to estimate FRET/cGMP changes within moving structures such as the vessel wall during vasodilation. The methods presented herein should be useful to visualize cGMP or other biochemical signals that are detectable with FRET-based biosensors, such as cyclic adenosine monophosphate or Ca2+, and to correlate them with respective vascular responses. With further refinement and combination of transgenic mouse models and intravital imaging technologies, we envision an exciting future, in which we are able to “watch” biochemistry, (patho-)physiology, and pharmacotherapy in the context of a living mammalian organism

Abstracts from the 8th International Conference on cGMP Generators, Effectors and Therapeutic Implications

This work was supported by a restricted research grant of Bayer AG

Expression of Connexin43 stimulates endothelial angiogenesis independently of gap junctional communication in vitro

Connexins (Cx) form gap junctions (GJ) and allow for intercellular communication. However, these proteins also modulate gene expression, growth, and cell migration. The downregulation of Cx43 impairs endothelial cell migration and angiogenetic potential. Conversely, endothelial Cx43 expression is upregulated in an in vivo angiogenesis model relying on hemodynamic forces. We studied the effects of Cx43 expression on tube formation and proliferation in HUVECs and examined its dependency on GJ communication. Expectedly, intercellular communication assessed by dye transfer was linked to Cx43 expression levels in HUVECs and was sensitive to a GJ blockade by the Cx43 mimetic peptide Gap27. The proliferation of HUVECs was not affected by Cx43 overexpression using Cx43 cDNA transfection, siRNA-mediated knockdown of Cx43, or the inhibition of GJ compared to the controls (transfection of an empty vector, scrambled siRNA, and the solvent). In contrast, endothelial tube and sprout formation in HUVECs was minimized after Cx43 knockdown and significantly enhanced after Cx43 overexpression. This was not affected by a GJ blockade (Gap27). We conclude that Cx43 expression positively modulates the angiogenic potential of endothelial cells independent of GJ communication. Since proliferation remained unaffected, we suggest that Cx43 protein may modulate endothelial cell migration, thereby supporting angiogenesis. The modulation of Cx43 expression may represent an exploitable principle for angiogenesis induction in clinical therapy

Activation of KCa3.1 by SKA-31 induces arteriolar dilatation and lowers blood pressure in normo- and hypertensive connexin40-deficient mice.

Background and purposeThe calcium-activated potassium channel KCa3.1 is expressed in the vascular endothelium where its activation causes endothelial hyperpolarization and initiates endothelium-derived hyperpolarization (EDH)-dependent dilatation. Here, we investigated whether pharmacological activation of KCa3.1 dilates skeletal muscle arterioles and whether myoendothelial gap junctions formed by connexin40 (Cx40) are required for EDH-type dilatations and pressure depressor responses in vivo.Experimental approachWe performed intravital microscopy in the cremaster muscle microcirculation and blood pressure telemetry in Cx40-deficient mice.Key resultsIn wild-type mice, the KCa3.1-activator SKA-31 induced pronounced concentration-dependent arteriolar EDH-type dilatations, amounting to ∼40% of maximal dilatation, and enhanced the effects of ACh. These responses were absent in mice devoid of KCa3.1 channels. In contrast, SKA-31-induced dilatations were not attenuated in mice with endothelial cells deficient in Cx40 (Cx40(fl/fl):Tie2-Cre). In isolated endothelial cell clusters, SKA-31 induced hyperpolarizations of similar magnitudes (by ∼38 mV) in Cx40(fl/fl):Tie2-Cre, ubiquitous Cx40-deficient mice (Cx40(-/-)) and controls (Cx40(fl/fl)), which were reversed by the specific KCa3.1-blocker TRAM-34. In normotensive wild-type and Cx40(fl/fl):Tie2-Cre as well as in hypertensive Cx40(-/-) animals, i.p. injections of SKA-31 (30 and 100 mg·kg(-1)) decreased arterial pressure by ∼32 mmHg in all genotypes. The depressor response to 100 mg·kg(-1) SKA-31 was associated with a decrease in heart rate.Conclusions and implicationsWe conclude that endothelial hyperpolarization evoked by pharmacological activation of KCa3.1 channels induces EDH-type arteriolar dilatations that are independent of endothelial Cx40 and Cx40-containing myoendothelial gap junctions. As SKA-31 reduced blood pressure in hypertensive Cx40-deficient mice, KCa3.1 activators may be useful drugs for severe treatment-resistant hypertension

Publisher Correction: A shear-dependent NO-cGMP-cGKI cascade in platelets acts as an auto-regulatory brake of thrombosis

The original version of this Article contained an error in the description of Supplementary Movie 4, in which the final sentence was inadvertently truncated. The HTML has been updated to include a corrected version of the ‘Description of Additional Supplementary Files’ file

Amplification of EDHF-type vasodilatations in TRPC1-deficient mice

BACKGROUND AND PURPOSE: TRPC1 channels are expressed in the vasculature and are putative candidates for intracellular Ca(2+) handling. However, little is known about their role in endothelium-dependent vasodilatations including endothelium-derived hyperpolarizing factor (EDHF) vasodilatations, which require activation of Ca(2+)-activated K(+) channels (K(Ca)). To provide molecular information on the role of TRPC1 for K(Ca) function and the EDHF signalling complex, we examined endothelium-dependent and independent vasodilatations, K(Ca) currents and smooth muscle contractility in TRPC1-deficient mice (TRPC1-/-). EXPERIMENTAL APPROACH: Vascular responses were studied using pressure/wire myography and intravital microscopy. We performed electrophysiological measurements, and confocal Ca(2+) imaging for studying K(Ca) channel functions and Ca(2+) sparks. KEY RESULTS: TRPC1 deficiency in carotid arteries produced a twofold augmentation of TRAM-34- and UCL1684-sensitive EDHF-type vasodilatations and of endothelial hyperpolarization to acetylcholine. NO-mediated vasodilatations were unchanged. TRPC1-/- exhibited enhanced EDHF-type vasodilatations in resistance-sized arterioles in vivo associated with reduced spontaneous tone. Endothelial IK(Ca)/SK(Ca)-type K(Ca) currents, smooth muscle cell Ca(2+) sparks and associated BK(Ca)-mediated spontaneous transient outward currents were unchanged in TRPC1-/-. Smooth muscle contractility induced by receptor-operated Ca(2+) influx or Ca(2+) release and endothelium-independent vasodilatations were unaltered in TRPC1-/-. TRPC1-/- exhibited lower systolic blood pressure as determined by tail-cuff blood pressure measurements. CONCLUSIONS AND IMPLICATIONS: Our data demonstrate that TRPC1 acts as a negative regulator of endothelial K(Ca) channel-dependent EDHF-type vasodilatations and thereby contributes to blood pressure regulation. Thus, we propose a specific role of TRPC1 in the EDHF–K(Ca) signalling complex and suggest that pharmacological inhibition of TRPC1, by enhancing EDHF vasodilatations, may be a novel strategy for lowering blood pressure

A shear-dependent NO-cGMP-cGKI cascade in platelets acts as an auto-regulatory brake of thrombosis

Nitric oxide (NO) inhibits thrombosis in part by stimulating cyclic guanosine monophosphate (cGMP) production and cGMP-dependent protein kinase I (cGKI) activity in platelets. Here, Wen et al. develop a cGMP sensor mouse to follow cGMP dynamics in platelets, and find that shear stress activates NO-cGMP-cGKI signaling during platelet aggregation to limit thrombosis

ADAMTS-7 Inhibits Re-endothelialization of Injured Arteries and Promotes Vascular Remodeling Through Cleavage of Thrombospondin-1

Background-ADAMTS-7, a member of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, was recently identified to be significantly associated genomewide with coronary artery disease. However, the mechanisms that link ADAMTS-7 and coronary artery disease risk remain elusive. We have previously demonstrated that ADAMTS-7 promotes vascular smooth muscle cell migration and postinjury neointima formation via degradation of a matrix protein cartilage oligomeric matrix protein. Because delayed endothelium repair renders neointima and atherosclerosis plaque formation after vessel injury, we examined whether ADAMTS-7 also inhibits re-endothelialization. Methods and Results-Wire injury of the carotid artery and Evans blue staining were performed in Adamts7(-/-) and wild-type mice. Adamts-7 deficiency greatly promoted re-endothelialization at 3, 5, and 7 days after injury. Consequently, Adamts-7 deficiency substantially ameliorated neointima formation in mice at days 14 and 28 after injury in comparison with the wild type. In vitro studies further indicated that ADAMTS-7 inhibited both endothelial cell proliferation and migration. Surprisingly, cartilage oligomeric matrix protein deficiency did not affect endothelial cell proliferation/migration and re-endothelialization in mice. In a further examination of other potential vascular substrates of ADAMTS-7, a labelfree liquid chromatography-tandem mass spectrometry secretome analysis revealed thrombospondin-1 as a potential ADAMTS-7 target. The subsequent studies showed that ADAMTS-7 was directly associated with thrombospondin-1 by its C terminus and degraded thrombospondin-1 in vivo and in vitro. The inhibitory effect of ADAMTS-7 on postinjury endothelium recovery was circumvented in Tsp1(-/-) mice. Conclusions-Our study revealed a novel mechanism by which ADAMTS-7 affects neointima formation. Thus, ADAMTS-7 is a promising treatment target for postinjury vascular intima hyperplasia.Cardiac & Cardiovascular SystemsPeripheral Vascular DiseaseSCI(E)[email protected]; [email protected]+13