Cor de Wit

Impaired Conduction of Vasodilation Along Arterioles in Connexin40-Deficient Mice

Connexins have been hypothesized to play an important role in intercellular communication within the vascular wall and may provide a mechanistic explanation for conduction of vasomotor responses. To test this hypothesis, we studied the transmission of vasomotor responses in the intact skeletal muscle microcirculation of connexin40-deficient mice (Cx40(-/-)). Arterioles were locally stimulated with hyperpolarizing dilators (acetylcholine [ACh] as well as bradykinin [Bk]) or depolarizing K(+) solution, and the resulting changes in diameter were measured using a videomicroscopy technique at the site of application and up to 1.32 mm upstream. Arterial pressure was elevated 25% in Cx40(-/-) mice (94+/-5 versus 75+/-4 mm Hg). Vessels selected for study had equivalent basal diameter and vasomotor tone in both genotypes of mice. Vasomotion was present in small arterioles of both genotypes, but its intensity was exaggerated in Cx40(-/-) mice. ACh and Bk induced dilation (33% and 53%, respectively, of maximal response) at the site of application that was of similar magnitude in both genotypes. These dilations were observed to spread upstream within <1 second without significant attenuation in Cx40(+/+) mice. However, spreading was severely attenuated in Cx40(-/-) animals (11+/-4% versus 35+/-7% with ACh and 38+/-5% versus 60+/-7% with Bk in Cx40(-/-) and Cx40(+/+), respectively; P<0.05). In contrast, conducted vasoconstrictions, induced by K(+) solution decreased equally with distance in both genotypes. These results support a significant role for Cx40 in vascular intercellular communication. Our observations indicate that Cx40 is required for normal transmission of endothelium-dependent vasodilator responses and may underlie altered vasomotion patterns.

Impaired Endothelium-Derived Hyperpolarizing Factor-Mediated Dilations and Increased Blood Pressure in Mice Deficient of the Intermediate-Conductance Ca2+-Activated K+ Channel

The endothelium plays a key role in the control of vascular tone and alteration in endothelial cell function contributes to several cardiovascular disease states. Endothelium-dependent dilation is mediated by NO, prostacyclin, and an endothelium-derived hyperpolarizing factor (EDHF). EDHF signaling is thought to be initiated by activation of endothelial Ca2+-activated K+ channels (KCa), leading to hyperpolarization of the endothelium and subsequently to hyperpolarization and relaxation of vascular smooth muscle. In the present study, we tested the functional role of the endothelial intermediate-conductance KCa (IKCa/KCa3.1) in endothelial hyperpolarization, in EDHF-mediated dilation, and in the control of arterial pressure by targeted deletion of KCa3.1. KCa3.1-deficient mice (KCa3.1−/−) were generated by conventional gene-targeting strategies. Endothelial KCa currents and EDHF-mediated dilations were characterized by patch-clamp analysis, myography and intravital microscopy. Disruption of the KCa3.1 gene abolished endothelial KCa3.1 currents and significantly diminished overall current through KCa channels. As a consequence, endothelial and smooth muscle hyperpolarization in response to acetylcholine was reduced in KCa3.1−/− mice. Acetylcholine-induced dilations were impaired in the carotid artery and in resistance vessels because of a substantial reduction of EDHF-mediated dilation in KCa3.1−/− mice. Moreover, the loss of KCa3.1 led to a significant increase in arterial blood pressure and to mild left ventricular hypertrophy. These results indicate that the endothelial KCa3.1 is a fundamental determinant of endothelial hyperpolarization and EDHF signaling and, thereby, a crucial determinant in the control of vascular tone and overall circulatory regulation.

Ischaemic heart disease in women: are there sex differences in pathophysiology and risk factors?Position Paper from the Working Group on Coronary Pathophysiology and Microcirculation of the European Society of Cardiology

Cardiovascular disease (CVD) is the leading cause of death in women, and knowledge of the clinical consequences of atherosclerosis and CVD in women has grown tremendously over the past 20 years. Research efforts have increased and many reports on various aspects of ischaemic heart disease (IHD) in women have been published highlighting sex differences in pathophysiology, presentation, and treatment of IHD. Data, however, remain limited. A description of the state of the science, with recognition of the shortcomings of current data, is necessary to guide future research and move the field forward. In this report, we identify gaps in existing literature and make recommendations for future research. Women largely share similar cardiovascular risk factors for IHD with men; however, women with suspected or confirmed IHD have less coronary atherosclerosis than men, even though they are older and have more cardiovascular risk factors than men. Coronary endothelial dysfunction and microvascular disease have been proposed as important determinants in the aetiology and prognosis of IHD in women, but research is limited on whether sex differences in these mechanisms truly exist. Differences in the epidemiology of IHD between women and men remain largely unexplained, as we are still unable to explain why women are protected towards IHD until older age compared with men. Eventually, a better understanding of these processes and mechanisms may improve the prevention and the clinical management of IHD in women.

Genetic deficit of SK3 and IK1 channels disrupts the endothelium-derived hyperpolarizing factor vasodilator pathway and causes hypertension.

Background— It has been proposed that activation of endothelial SK3 (KCa2.3) and IK1 (KCa3.1) K+ channels plays a role in the arteriolar dilation attributed to an endothelium-derived hyperpolarizing factor (EDHF). However, our understanding of the precise function of SK3 and IK1 in the EDHF dilator response and in blood pressure control remains incomplete. To clarify the roles of SK3 and IK1 channels in the EDHF dilator response and their contribution to blood pressure control in vivo, we generated mice deficient for both channels. Methods and Results— Expression and function of endothelial SK3 and IK1 in IK1−/−/SK3T/T mice was characterized by patch-clamp, membrane potential measurements, pressure myography, and intravital microscopy. Blood pressure was measured in conscious mice by telemetry. Combined IK1/SK3 deficiency in IK1−/−/SK3T/T (+doxycycline) mice abolished endothelial KCa currents and impaired acetylcholine-induced smooth muscle hyperpolarization and EDHF-mediated dilation in conduit arteries and in resistance arterioles in vivo. IK1 deficiency had a severe impact on acetylcholine-induced EDHF-mediated vasodilation, whereas SK3 deficiency impaired NO-mediated dilation to acetylcholine and to shear stress stimulation. As a consequence, SK3/IK1-deficient mice exhibited an elevated arterial blood pressure, which was most prominent during physical activity. Overexpression of SK3 in IK1−/−/SK3T/T mice partially restored EDHF- and nitric oxide–mediated vasodilation and lowered elevated blood pressure. The IK1-opener SKA-31 enhanced EDHF-mediated vasodilation and lowered blood pressure in SK3-deficient IK1+/+/SK3T/T (+doxycycline) mice to normotensive levels. Conclusions— Our study demonstrates that endothelial SK3 and IK1 channels have distinct stimulus-dependent functions, are major players in the EDHF pathway, and significantly contribute to arterial blood pressure regulation. Endothelial KCa channels may represent novel therapeutic targets for the treatment of hypertension.

Connexin40 is essential for the pressure control of renin synthesis and secretion.

Renin secretion and synthesis in renal juxtaglomerular cells are controlled by short feed back loops involving angiotensin II and the intrarenal blood pressure. The operating mechanisms of these negative feed back regulators are widely unknown, except for the fact that both require calcium to exert their inhibitory action. We here show that in the absence of connexin40 (Cx40), which form gap junctions between juxtaglomerular and endothelial cells, the negative control of renin secretion and synthesis by angiotensin II and by intravasal pressure is abrogated, while the regulation by salt intake and &bgr;-adrenergic stimulation is maintained. Renin secretion from Cx40-deficient kidneys or wild-type kidneys treated with the nonselective gap junction blocker 18&agr;-glycyrrhetinic acid (10 &mgr;mol/L) resembles the situation in wild-type kidneys in the absence of extracellular calcium. This disturbed regulation is reflected by an enhanced plasma renin concentration despite an elevated blood pressure in Cx40-deficient mice. These findings indicate that Cx40 connexins and likely intercellular communication via Cx40-dependent gap junctions mediate the calcium-dependent inhibitor effects of angiotensin II and of intrarenal pressure on renin secretion and synthesis. Because Cx40 gap junctions are also formed between renin producing cells and endothelial cells our finding could provide additional information to suggest that the endothelium may be strongly involved in the control of the renin system.

Endothelium‐specific replacement of the connexin43 coding region by a lacZ reporter gene

Summary: The murine gap junction protein connexin43 (Cx43) is expressed in blood vessels, with vastly different contribution by endothelial and smooth muscle cells. We have used the Cre recombinase under control of TIE2 transcriptional elements to inactivate a floxed Cx43 gene specifically in endothelial cells. Cre‐mediated deletion led to replacement of the Cx43 coding region by a lacZ reporter gene. This allowed us to monitor the extent of deletion and to visualize the endothelial expression pattern of Cx43. We found widespread endothelial expression of the Cx43 gene during embryonic development, which became restricted largely to capillaries and small vessels in all adult organs examined. Mice lacking Cx43 in endothelium did not exhibit altered blood pressure, in contrast to mice deficient in Cx40. Our results show that lacZ activation after deletion of the target gene allows us to determine the extent of cell type‐specific deletion after phenotypical investigation of the same animal. genesis 29:1–13, 2001.

Magnetofection--a highly efficient tool for antisense oligonucleotide delivery in vitro and in vivo.

Delivery of antisense oligodesoxynucleotides (ODN) into primary cells is a specific strategy for research with therapeutic perspectives but transfection-associated difficulties. We established the technique of magnetofection to enhance ODN delivery at low toxicity and procedure time in vitro and in vivo. In vitro, target knockout was assessed at protein and mRNA levels and by measuring superoxide generation after antisense magnetofection against the p22(phox) subunit of endothelial NAD(P)H-oxidase. Under magnetic field guidance, low-dose magnetic particle-bound ODN were transfected to 84% human umbilical vein endothelial cells within 15 min followed by nuclear accumulation within 2 h, which required 24 h using standard methods. Antisense magnetofection against p22(phox) significantly decreased basal and prevented stimulated superoxide release due to loss of NAD(P)H-oxidase activity by mRNA knockout as assessed after 24 h. Knockout of endothelial phosphatase SHP-1 and connexin 37 proteins confirmed the methods efficiency. Transfection-associated toxicity was minimal. Twenty-four hours after injection of fluorescence-labeled ODN into femoral arteries of male mice, there was specific ODN uptake only into cremaster vessels exposed to magnetic fields during injection. Magnetofection is an ideal tool for delivery of functionally active ODN to difficult-to-transfect cells to study gene/protein function and a promising strategy for targeted ODN delivery in vivo.

Dysfunctional nitric oxide signalling increases risk of myocardial infarction

Myocardial infarction, a leading cause of death in the Western world, usually occurs when the fibrous cap overlying an atherosclerotic plaque in a coronary artery ruptures. The resulting exposure of blood to the atherosclerotic material then triggers thrombus formation, which occludes the artery. The importance of genetic predisposition to coronary artery disease and myocardial infarction is best documented by the predictive value of a positive family history. Next-generation sequencing in families with several affected individuals has revolutionized mutation identification. Here we report the segregation of two private, heterozygous mutations in two functionally related genes, GUCY1A3 (p.Leu163Phefs*24) and CCT7 (p.Ser525Leu), in an extended myocardial infarction family. GUCY1A3 encodes the α1 subunit of soluble guanylyl cyclase (α1-sGC), and CCT7 encodes CCTη, a member of the tailless complex polypeptide 1 ring complex, which, among other functions, stabilizes soluble guanylyl cyclase. After stimulation with nitric oxide, soluble guanylyl cyclase generates cGMP, which induces vasodilation and inhibits platelet activation. We demonstrate in vitro that mutations in both GUCY1A3 and CCT7 severely reduce α1-sGC as well as β1-sGC protein content, and impair soluble guanylyl cyclase activity. Moreover, platelets from digenic mutation carriers contained less soluble guanylyl cyclase protein and consequently displayed reduced nitric-oxide-induced cGMP formation. Mice deficient in α1-sGC protein displayed accelerated thrombus formation in the microcirculation after local trauma. Starting with a severely affected family, we have identified a link between impaired soluble-guanylyl-cyclase-dependent nitric oxide signalling and myocardial infarction risk, possibly through accelerated thrombus formation. Reversing this defect may provide a new therapeutic target for reducing the risk of myocardial infarction.

Connexins and gap junctions in the EDHF phenomenon and conducted vasomotor responses

It is becoming increasingly evident that electrical signaling via gap junctions plays a central role in the physiological control of vascular tone via two related mechanisms (1) the endothelium-derived hyperpolarizing factor (EDHF) phenomenon, in which radial transmission of hyperpolarization from the endothelium to subjacent smooth muscle promotes relaxation, and (2) responses that propagate longitudinally, in which electrical signaling within the intimal and medial layers of the arteriolar wall orchestrates mechanical behavior over biologically large distances. In the EDHF phenomenon, the transmitted endothelial hyperpolarization is initiated by the activation of Ca2+-activated K+ channels channels by InsP3-induced Ca2+ release from the endoplasmic reticulum and/or store-operated Ca2+ entry triggered by the depletion of such stores. Pharmacological inhibitors of direct cell-cell coupling may thus attenuate EDHF-type smooth muscle hyperpolarizations and relaxations, confirming the participation of electrotonic signaling via myoendothelial and homocellular smooth muscle gap junctions. In contrast to isolated vessels, surprisingly little experimental evidence argues in favor of myoendothelial coupling acting as the EDHF mechanism in arterioles in vivo. However, it now seems established that the endothelium plays the leading role in the spatial propagation of arteriolar responses and that these involve poorly understood regenerative mechanisms. The present review will focus on the complex interactions between the diverse cellular signaling mechanisms that contribute to these phenomena.

Nitric Oxide-Induced Decrease in Calcium Sensitivity of Resistance Arteries Is Attributable to Activation of the Myosin Light Chain Phosphatase and Antagonized by the RhoA/Rho Kinase Pathway

Background NO‐induced dilations in resistance arteries (RAs) are not associated with decreases in vascular smooth muscle cell Ca2+. We tested whether a cGMP‐dependent activation of the smooth muscle myosin light chain phosphatase (MLCP) resulting in a Ca2+ desensitization of the contractile apparatus was the underlying mechanism and whether it could be antagonized by the RhoA pathway. Methods and Results The Ca2+ sensitivity of RA was assessed as the relation between changes in diameter and [Ca2+], in depolarized RA (120 mol/L K+) exposed to stepwise increases in Ca2+ex (0 to 3 mmol/L). Effects of 10 μmol/L sodium nitroprusside (SNP) on Ca2+ sensitivity were determined before and after application of the soluble guanylate cyclase inhibitor ODQ (1 μmol/L) and the MLCP inhibitor calyculin A (120 nmol/L) and in presence of the RhoA‐activating phospholipid sphingosine‐1‐phosphate (S1P, 12 nmol/L). SNP‐induced dilations were also studied in controls and in RAs pretreated with the Rho kinase inhibitor Y27632 or transfected with a dominant‐negative RhoA mutant (N19RhoA). Constrictions elicited by increasing Ca2+ex were significantly attenuated by SNP, which, however, left associated increases in [Ca2+ unaffected. This NO‐induced attenuation was blocked by ODQ, calyculin A, and S1P. The S1P‐induced translocation of RhoA indicating activation of the GTPase was not reversed by SNP. Inhibition of RhoA/Rho kinase by N19RhoA or Y27632 significantly augmented SNP‐induced dilations. Conclusions NO dilates RA by activating the MLCP in a cGMP‐dependent manner, thereby reducing the apparent Ca2+ sensitivity of the contractile apparatus. MLCP inactivation via the RhoA/Rho kinase pathway antagonizes this Ca2+‐desensitizing effect that, in turn, can be restored using RhoA/Rho kinase inhibitors. (Circulation. 2003;107:3081‐3087.)