Endothelium Derived Autacoids

The potential role of endothelial cells in flow-dependent dilation gained considerable interest after the pioneering observation by Furchgott and Zawadzki [1] that the endothelium can actively induce changes in vascular tone by the release of a labile relaxing factor. In fact, an obligatory role of endothelial cells in sensing changes in blood flow signals and transducing them into vasodilator responses was demonstrated in large conduit arteries in situ as well as in arteries in vitro. Furthermore, clinical studies revealed that the flow-dependent dilation, which occurs in different vascular beds in humans, is reduced or even abolished by hypercholesterolemia and arteriosclerosis. Although there are a number of additional endothelium-derived vasodilator and vasoconstrictor autacoids [endothe-lin-1, prostacyclin (PGI2), prostaglandin H2 the superoxide anion (O2-), and the endothelium-derived hyperpolarizing factor: EDHF], none of these autacoids play as central a role in the regulation of vascular tone and homeostasis as the free radical nitric oxide (NO).

Nitric Oxide

In endothelial cells NO is generated from the amino acid L-arginine by the endothelial NO synthase (eNOS), which is a constitutively expressed membrane-bound enzyme. Functional eNOS is a homodimer whose activity is determined by its interaction with a group of eNOS-associated proteins that include calmodulin, caveolin-1, and heat shock protein 90 (hsp90), as well as by intracellular levels of Ca2+ ([Ca2+]j), and the phosphorylation of several regulatory serine and threonine residues.

Distinct signaling pathways modulate the activity of eNOS in response to agonists and hemodynamic stimulation. Whereas the former is largely a Ca2+-dependent process, the activation of eNOS by fluid shear stress does not require a maintained increase in [Ca2+] but is dependent on the activation of the phosphatidylinositol 3-kinase and the subsequent activation of the serine kinase Akt, which phosphorylates eNOS on Ser1177 (Figure 1). This step increases enzyme activity two- to four-fold and thus increases NO production. Provided the stimulus is maintained, Akt remains activated for several hours, as does the production of NO. The NO-dependent relaxation of vascular smooth muscle cells involves the activation of the soluble guanylyl cyclase and the subsequent increase in cyclic GMP levels, which, via the activation of G kinase, elicits a decrease in vascular smooth muscle cell [Ca2+]r

NO is, however much more than a vasodilator and is generally thought to play a central role in maintaining the endothelium in a noninflammatory phenotype by influencing the activity of several transcription factors and thus attenuating the expression of proinflammatory genes such as the adhesion molecules E-selectin and vascular cell adhesion molecule-1 (VCAM-1). With the progression of vascular disease, NO-dependent vasodilatation becomes impaired, partly as a consequence of the interaction of NO with O2- generated by enzymes such as NADPH oxidase, to generate the strong oxidant peroxynitrite (ONOO-) and partly as a consequence of eNOS uncoupling. The latter phenomenon relates to a switch from the production of NO to the generation of O2- by eNOS and has been linked to decreased cellular levels of the essential cofactor tetra-hydrobiopterin. The consequence of these changes is a decrease in the bioavailability of NO and a reduction in the vasodilator response to agonists such as acetylcholine as well as the activation of redox-sensitive transcription factors (e.g., nuclear factor kappa B [NF-kB]), the activation of which is normally suppressed by NO.

Prostacyclin

Cyclo-oxygenase (COX) is the key enzyme in the synthesis of prostanoids, and two COX isoforms (COX1 and COX-2) have been identified in endothelial cells. Although these enzymes catalyze the same reaction, they have distinct pharmacological profiles and biological roles. COX-1 is constitutively expressed in endothelial cells, whereas COX-2 is expressed in other cell types in response to proinflam-matory stimuli. The availability of the COX substrate, arachidonic acid, which is released from membrane phospholipids by phospholipase A2 (PLA2), is the rate-limiting step in prostaglandin synthesis, and PGI2 is the major product of the subsequent COX pathway in all arterial and venous endothelial cells studied so far. As a consequence of the Ca2+ sensitivity of PLA2, the synthesis of PGI2 by endothelial cells is a Ca2+-dependent process. This codepen-dency on Ca2+ means that in many circumstances NO, PGI2, and EDHF can be generated simultaneously.

The apparent redundancy in the major vasoactive autacoid generating pathways means that the loss of one vasodilator can be compensated for by one of the others. For

Shear-*

stress-*

Figure 1 Endothelial cell activation by fluid shear stress increases eNOS phosphorylation and activity. In unstimulated endothelial cells the eNOS dimer is associated with caveolin, which inhibits its activation, which means that NO output is low (A). In response to the application of fluid shear stress, the phosphatidylinositol 3-kinase (PI 3-K) is activated and in turn activates Akt, which associates with the eNOS scaffolding protein hsp90 and phosphorylates eNOS on Ser1177 (B). This step elicits the dissociation from caveolin and increases NO production. Shear stress-induced increases in endothelial NO production can be abrogated by preventing the activation of PI 3-K (C). (see color insert)

Figure 1 Endothelial cell activation by fluid shear stress increases eNOS phosphorylation and activity. In unstimulated endothelial cells the eNOS dimer is associated with caveolin, which inhibits its activation, which means that NO output is low (A). In response to the application of fluid shear stress, the phosphatidylinositol 3-kinase (PI 3-K) is activated and in turn activates Akt, which associates with the eNOS scaffolding protein hsp90 and phosphorylates eNOS on Ser1177 (B). This step elicits the dissociation from caveolin and increases NO production. Shear stress-induced increases in endothelial NO production can be abrogated by preventing the activation of PI 3-K (C). (see color insert)

example, in wild-type mice flow-dependent vasodilatation in gracilis muscle arterioles appears to be mediated by NO. However, in male eNOS-/- mice, flow-induced vasodilatation of these arterioles can be attributed to the generation of PGI2.

The Endothelium-Derived Hyperpolarizing Factor

In various blood vessels, endothelium-dependent relaxations can be accompanied by the endothelium-dependent hyperpolarization of smooth muscle cells. These endothelium-dependent relaxations and hyperpolarizations can be partially or totally resistant to inhibitors of COX and NO synthases and could be observed without an increase in intracellular levels of cyclic nucleotides in the smooth muscle cells. Therefore the existence of an additional pathway that involves smooth muscle hyperpolarization was suggested and attributed to an endothelium-derived hyperpolar-izing factor. The contribution of EDHF-mediated responses as a mechanism for endothelium-dependent relaxation increases as the vessel size decreases, apart from the coronary and renal vascular beds in which EDHF also plays a major role in conduit arteries. Different mechanisms appear to underlie EDHF-mediated responses in different arteries; however, experimental evidence favors three explanations for the EDHF phenomenon (Figure 2):

1. K+ ions are released from stimulated endothelial cells through Ca2+-activated K+ (KCa) channels and subsequently elicit relaxation by activating K+ channels and/or the Na+/K+-ATPase on vascular smooth muscle cells, thus inducing hyperpolarization and decreasing the open probability of voltage-operated Ca2+ channels, through which the Ca2+ required for contraction enters the cell.

2. An increase in endothelial [Ca2+] triggers the activation of a phospholipase A2 that liberates arachidonic acid from membrane phospholipids. The arachidonic acid is then metabolized by cytochrome P450 epoxygenase(s) to epoxyeicosatrienoic acids (EETs), which either contribute to the hyperpolarization of endothelial cells or are released from endothelial cells to activate K+Ca channels on underlying vascular smooth muscle cells.

3. Direct transfer of an electrical signal (i.e., endothelial cell hyperpolarization) to vascular smooth muscle cells via myo-endothelial gap junctions. This mechanism appears to be particularly important in the microcirculation.

The majority of the work performed to date that has led to the characterization of EDHF-mediated responses has involved the use of receptor-dependent agonists such as acetylcholine and bradykinin. However, the limited studies that have addressed the response to hemodynamic stimuli suggests that pulsatile stretch is a particularly important stimulus for the generation of EDHF. This phenomenon may be related to the fact that pulsatile stretch elicits much larger and more prolonged increases in [Ca2+] in endothelial cells than fluid shear stress does. Indeed, KCa channel-dependent hyperpolarization is preferentially stimulated by pulsatile flow as opposed to steady flow, and pulsatile stretch/cyclic strain has been reported to increase the generation of EETs by native porcine coronary endothelial cells as well as to increase the expression of the EET-generating cytochrome P450 epoxygenase.

Figure 2 Three models contributing to endothelium-derived hyperpolarizing factor-mediated relaxation. (A) The K+ ion hypothesis. In response to the receptor (R)-dependent activation of endothelial cells (EC), intracellular concentration of Ca2+ ([Ca2+]j) increases and activate Ca2+-dependent K+ (K+Ca) channels, which results in the efflux of K+ ions into the subintimal space and endothelial cell hyperpolarization (lightning symbol). The increase in extracellular K+ activates inwardly rectifying K+ channels (K+IR) and/or the Na-K-ATPase which elicit vascular smooth muscle cell (VSMC) hyperpolarization and decrease the open probability of voltage-dependent Ca2+ (VOC) channels. As a consequence [Ca2+] in the smooth muscle cells decreases and relaxation occurs. (B) The cytochrome P450 hypothesis. An increase in [Ca2+] activates a phospholipase A2, which liberates arachidonic acid (AA) from membrane lipids. Arachidonic acid is then metabolized to epoxyeicosatrienoic acids (EETs) by a cytochrome P450 2C (CYP 2C) epoxygenase. EETs then increase the open probability of K+Ca in the underlying smooth muscle cells to elicit hyperpolarization and relaxation. (C) The gap junction hypothesis. Endothelial cell activation as described elicits endothelial cell hyperpolarization by activating K+Ca channels. This hyperpolarization is, however, transferred to smooth muscle cells via myo-endothelial gap junctions rather than by the release of an endothelium-derived factor. The final steps are the same, that is, inhibition of Ca2+ entry into smooth muscle cells and relaxation. (see color insert)

Figure 2 Three models contributing to endothelium-derived hyperpolarizing factor-mediated relaxation. (A) The K+ ion hypothesis. In response to the receptor (R)-dependent activation of endothelial cells (EC), intracellular concentration of Ca2+ ([Ca2+]j) increases and activate Ca2+-dependent K+ (K+Ca) channels, which results in the efflux of K+ ions into the subintimal space and endothelial cell hyperpolarization (lightning symbol). The increase in extracellular K+ activates inwardly rectifying K+ channels (K+IR) and/or the Na-K-ATPase which elicit vascular smooth muscle cell (VSMC) hyperpolarization and decrease the open probability of voltage-dependent Ca2+ (VOC) channels. As a consequence [Ca2+] in the smooth muscle cells decreases and relaxation occurs. (B) The cytochrome P450 hypothesis. An increase in [Ca2+] activates a phospholipase A2, which liberates arachidonic acid (AA) from membrane lipids. Arachidonic acid is then metabolized to epoxyeicosatrienoic acids (EETs) by a cytochrome P450 2C (CYP 2C) epoxygenase. EETs then increase the open probability of K+Ca in the underlying smooth muscle cells to elicit hyperpolarization and relaxation. (C) The gap junction hypothesis. Endothelial cell activation as described elicits endothelial cell hyperpolarization by activating K+Ca channels. This hyperpolarization is, however, transferred to smooth muscle cells via myo-endothelial gap junctions rather than by the release of an endothelium-derived factor. The final steps are the same, that is, inhibition of Ca2+ entry into smooth muscle cells and relaxation. (see color insert)

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