Kjestine Schmidt

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 label-free 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.

Defective Cx40 Maintains Cx37 Expression but Intact Cx40 Is Crucial for Conducted Dilations Irrespective of Hypertension

The gap junction channel protein connexin40 (Cx40) is crucial in vascular and renal physiology, because Cx40-deficient mice exhibit impaired conduction of endothelium-dependent dilations and pronounced hypertension. The latter precludes mechanistic insights into the role of endothelial Cx40, because long-lasting hypertension itself may affect conduction and Cx expression. We aimed to identify endothelial Cx40 functions, their dependency on the conductive capability, and to separate these from hypertension-related alterations. We assessed conduction and Cx expression in mice with cell type–specific deletion of Cx40 and in mice expressing a defective Cx40 (Cx40A96S) identified in humans, which forms nonconducting gap junction channels. Confined arteriolar stimulation with acetylcholine or bradykinin elicited local dilations that conducted upstream without attenuation of the amplitude for distances up to 1.2-mm in controls with a floxed Cx40 gene (Cx40fl/fl). Conducted responses in hypertensive animals devoid of Cx40 in renin-producing cells were unaltered but remote dilations were reduced in normotensive animals deficient for Cx40 in endothelial cells (Cx40fl/fl:Tie2-Cre). Surprisingly, Cx37 expression was undetectable by immunostaining in arteriolar endothelium only in Cx40fl/fl:Tie2-Cre; however, transcriptional activity of Cx37 in the cremaster was comparable with Cx40fl/fl controls. Cx40A96S mice were hypertensive with preserved expression of Cx40 and Cx37. Nevertheless, conducted responses were blunted. We conclude that endothelial Cx40 is necessary to support conducted dilations initiated by endothelial agonists and to locate Cx37 into the plasma membrane. These functions are unaltered by long-lasting hypertension. In the presence of a nonconducting Cx40, Cx37 is present but cannot support the conduction highlighting the importance of endothelial Cx40.

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

The 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.

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 Ca2+ handling. However, little is known about their role in endothelium‐dependent vasodilatations including endothelium‐derived hyperpolarizing factor (EDHF) vasodilatations, which require activation of Ca2+‐activated K+ channels (KCa). To provide molecular information on the role of TRPC1 for KCa function and the EDHF signalling complex, we examined endothelium‐dependent and independent vasodilatations, KCa currents and smooth muscle contractility in TRPC1‐deficient mice (TRPC1‐/‐).

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.

Keep calm and carry on: miR-1298 prevents up-regulation of Cx43 and secures a quiescent vascular smooth muscle cell

This editorial refers to ‘MicroRNA-1298 is regulated by DNA methylation and affects vascular smooth muscle cell function by targeting connexin 43’ by W. Hu et al. , pp. 534–545. Atherosclerosis constitutes the leading cause of cardiovascular disease resulting in ischaemia of dependent organs due to arterial occlusion. It affects not only heart and brain, but also limbs and other organs with fatal consequences for the individuum. The process invokes profound remodelling of the entire vascular wall driven by inflammation, which affects endothelial cells and promotes the activation of vascular smooth muscle cells (VSMCs). In healthy vessels, VSMCs retain a quiescent, contractile state but in response to environmental cues (chemical and mechanical) they adopt an activated, synthetic state characterized also by proliferation and migration. This remarkable plasticity is achieved through a substantial change in gene expression profile, which is likely governed by epigenetic and/or transcriptional control mechanisms. In addition to transcriptional regulation, non-coding, single-stranded, small RNAs (typically ∼22 nucleotides) contribute substantially to the phenotypic modulation of VSMCs through post-transcriptional regulation of gene expression (mRNA degradation or translational repression).1,2 These so-called microRNAs (miRNAs/miRs) promote or inhibit the switch of VSMCs to the synthetic state. For example, the miR-143/145 cluster supports a contractile state and was down-regulated by vascular injury, implicating that its lack fosters the synthetic state.3,4 Conversely, other miRNAs (miR-21, miR-221/222) were found to promote the synthetic …

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

Mechanisms that limit thrombosis are poorly defined. One of the few known endogenous platelet inhibitors is nitric oxide (NO). NO activates NO sensitive guanylyl cyclase (NO-GC) in platelets, resulting in an increase of cyclic guanosine monophosphate (cGMP). Here we show, using cGMP sensor mice to study spatiotemporal dynamics of platelet cGMP, that NO-induced cGMP production in pre-activated platelets is strongly shear-dependent. We delineate a new mode of platelet-inhibitory mechanotransduction via shear-activated NO-GC followed by cGMP synthesis, activation of cGMP-dependent protein kinase I (cGKI), and suppression of Ca2+ signaling. Correlative profiling of cGMP dynamics and thrombus formation in vivo indicates that high cGMP concentrations in shear-exposed platelets at the thrombus periphery limit thrombosis, primarily through facilitation of thrombus dissolution. We propose that an increase in shear stress during thrombus growth activates the NO-cGMP-cGKI pathway, which acts as an auto-regulatory brake to prevent vessel occlusion, while preserving wound closure under low shear.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.

Oxidant sensor in the cGMP-binding pocket of PKGIα regulates nitroxyl-mediated kinase activity

Despite the mechanisms for endogenous nitroxyl (HNO) production and action being incompletely understood, pharmacological donors show broad therapeutic promise and are in clinical trials. Mass spectrometry and site-directed mutagenesis showed that chemically distinct HNO donors 1-nitrosocyclohexyl acetate or Angeli’s salt induced disulfides within cGMP-dependent protein kinase I-alpha (PKGIα), an interdisulfide between Cys42 of the two identical subunits of the kinase and a previously unobserved intradisulfide between Cys117 and Cys195 in the high affinity cGMP-binding site. Kinase activity was monitored in cells transfected with wildtype (WT), Cys42Ser or Cys117/195Ser PKGIα that cannot form the inter- or intradisulfide, respectively. HNO enhanced WT kinase activity, an effect significantly attenuated in inter- or intradisulfide-deficient PKGIα. To investigate whether the intradisulfide modulates cGMP binding, real-time imaging was performed in vascular smooth muscle cells expressing a FRET-biosensor comprising the cGMP-binding sites of PKGIα. HNO induced FRET changes similar to those elicited by an increase of cGMP, suggesting that intradisulfide formation is associated with activation of PKGIα. Intradisulfide formation in PKGIα correlated with enhanced HNO-mediated vasorelaxation in mesenteric arteries in vitro and arteriolar dilation in vivo in mice. HNO induces intradisulfide formation in PKGIα, inducing the same effect as cGMP binding, namely kinase activation and thus vasorelaxation.

Real-time imaging of cGMP signals in platelets.

Background cGMP plays an essential role in platelet aggregation. However, the intracellular cGMP concentrations and the spatiotemporal dynamics of cGMP signals in platelets during hemostasis and thrombosis are largely unknown. To visualize cGMP signals in vivo, we have generated so-called cGMP sensor knock-in mice [1]. A Cre recombinaseactivatable expression cassette encoding the fluorescence resonance energy transfer (FRET)-based cGMP sensor, cGi500, driven by the CAG promoter was integrated into the Rosa26 locus. Depending on the strategy to activate sensor expression, these mice can show either ubiquitous or tissue-specific sensor expression allowing for delineation of cGMP signaling in live cells in vitro and in vivo.