SUMMARY Experiments were designed to investigate the importance of vascular endothelium in the vasomotor response to increases in flow as observed in conduit arteries (flow-dependent dilation). The diameter changes of femoral arteries (sonomicrometry) in response to increases in flow before and after endothelial damage procedures were studied in 23 dogs anesthetized with sodium pentobarbital. The functional integrity of the endothelial cells underneath the diameter sensors was tested by intraarterial acetylcholine (local acetylcholine dilation) applied proximally to the sensors while a constant flow was maintained. Unilateral augmentation of femoral arterial flow (4.6 ± 1.9-fold) induced by peripheral vasodilation or by arteriovenous shunt, elicited dilation (increase in diameter, 116 ± 91 fim) in 18 of 23 dogs, whereas the diameter of the contralateral control artery was not affected. Mechanical removal of the endothelial cells by means of a balloon catheter abolished both the flowdependent dilation and the local acetylcholine dilation, whereas the vasomotor responses to norepinephrine and nitroglycerin were not affected. Brief perfusions (1 minute) of the arteries with cell-free hydrogen peroxide solution (90 mM) also abolished the flow-dependent dilation and attenuated the local acetylcholine dilation (by 27 ± 19%; p < 0.02), while the responses to norepinephrine and nitroglycerin were not altered. These results suggest that endothelial cells act as mediators of flowdependent dilation. (Hypertension 8: 37-44, 1986) KEY WORDS • flow-dependent dilation • vascular endothelium • endothelium-mediated dilation • hydrogen peroxide T HE dilator response of conduit arteries to an augmentation of blood flow (flow-dependent dilation) was observed more than 50 years ago, 1 and flow-dependent dilation has been suggested as the principal response in a variety of physiological vascular adaptations, such as collateralization and long-term diameter adaptation to increased flow loads.2 However, the underlying mechanism of this vasodilation is still a matter of debate. Originally the vasodilation was thought to be elicited by a peripheral conducting mechanism, 13 but more recent experiments clearly indicate that a local mechanism is responsible. 4 This explanation implies that vascular wall structures sensitive to flow changes are involved.In view of the accumulating evidence for the modifying role of the vascular endothelium on vascular reactivity, and based on preliminary experiments in isolated arteries, we proposed that the vasodilation in response to flow increases is an endothelium-mediated reaction. 5 The goal of the present study was to demonstrate that the endothelium is functionally important in flow-dependent dilation under in vivo conditions.

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.

IntroductionPlatelet activation is thought to be a key event in acute vascular thrombosis. Therefore, prevention of enhanced platelet activation is a major target of therapeutic strategies fighting cardiovascular and cerebrovascular diseases. [1][2][3] An important stimulus for physiologic platelet activation and thrombus formation is the contact of platelets with components of the subendothelial matrix, like collagen. 4 Although Marcus et al have shown as early as 1977 that platelets have the ability to release superoxide anions (O 2 Ϫ ), 5 it was only recently proposed that platelets stimulated by collagen produce reactive oxygen species (ROS) such as hydrogen peroxide, 6 hydroxyl radicals, 7 or O 2 Ϫ . 7,8 While O 2 Ϫ , a highly reactive radical, damages cells in high concentrations by reacting with proteins, lipids, and DNA, in low concentrations its continuous production, with similarity to second messengers, has been suggested to indirectly affect signal transduction processes. 9,10 Platelet agonists other than collagen, such as thrombin or ADP, do not seem to induce ROS formation during aggregation. 8 This difference raises the question whether O 2 Ϫ formation could serve a modulating function when thrombus formation is induced by collagen.The cellular source of platelet O 2 Ϫ is unclear. Growing evidence supports the assumption that platelet activation by collagen is specifically due to binding to the glycoprotein VI (GPVI)-receptor, 11,12 resulting in a cascade of tyrosine phosphorylation events ultimately leading to activation of phospholipase C␥ (PLC␥), 13 which is known to strongly activate protein kinase C (PKC) through production of diacylglycerol. 4 Recently, evidence for the existence of a neutrophil-type reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase in platelets that can be activated by PKC and is involved in O 2 Ϫ formation has been presented, [14][15][16] similar as in other O 2 Ϫ -generating systems, like the vascular endothelium. In endothelial cells, an NAD(P)H oxidase is the main source of O 2 Ϫ . 17 As O 2 Ϫ readily reacts with NO, this has been suggested to result in attenuated 18,19 and the role of O 2 Ϫ in the regulation of vascular tone has become a major focus of interest. 17 Moreover, antioxidants like N-acetylcysteine (NAC) have been shown to exert direct antiaggregatory effects. 20 Although these findings raise the possibility that platelet-derived O 2 Ϫ is involved in regulating platelet activation, evidence for a role of platelet-derived O 2 Ϫ in platelet function is rare. In a canine model of coronary arterial thrombosis, thrombus formation was regulated by intraplatelet redox state. 21 Leo and colleagues have shown that platelets subjected to anoxia/ reoxygenation are more reactive, due to an enhanced O 2 Ϫ generation. 14 However, so far it remains unclear whether an enhanced O 2 Ϫ production occurs also during direct platelet activation, such as with collagen, and how this could affect thrombus growth. Whereas Supported by a grant from the Friedrich-...

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.

In vitro models incorporating the complexity and function of adult human tissues are highly desired for translational research. Whilst vital slices of human myocardium approach these demands, their rapid degeneration in tissue culture precludes long-term experimentation. Here, we report preservation of structure and performance of human myocardium under conditions of physiological preload, compliance, and continuous excitation. In biomimetic culture, tissue slices prepared from explanted failing human hearts attain a stable state of contractility that can be monitored for up to 4 months or 2000000 beats in vitro. Cultured myocardium undergoes particular alterations in biomechanics, structure, and mRNA expression. The suitability of the model for drug safety evaluation is exemplified by repeated assessment of refractory period that permits sensitive analysis of repolarization impairment induced by the multimodal hERG-inhibitor pentamidine. Biomimetic tissue culture will provide new opportunities to study drug targets, gene functions, and cellular plasticity in adult human myocardium.