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The endothelial nitric oxide synthase (eNOS), the expression of which is regulated by a range of transcriptional and posttranscriptional mechanisms, generates nitric oxide (NO) in response to a number of stimuli. The physiologically most important determinants for the continuous generation of NO and thus the regulation of local blood flow are fluid shear stress and pulsatile stretch. Although eNOS activity is coupled to changes in endothelial cell Ca(2+) levels, an increase in Ca(2+) alone is not sufficient to affect enzyme activity because the binding of calmodulin (CaM) and the flow of electrons from the reductase to the oxygenase domain of the enzyme is dependent on protein phosphorylation and dephosphorylation. Two amino acids seem to be particularly important in regulating eNOS activity and these are a serine residue in the reductase domain (Ser(1177)) and a threonine residue (Thr(495)) located within the CaM-binding domain. Simultaneous alterations in the phosphorylation of Ser(1177) and Thr(495) in response to a variety of stimuli are regulated by a number of kinases and phosphatases that continuously associate with and dissociate from the eNOS signaling complex. eNOS associated proteins, such as caveolin, heat shock protein 90, eNOS interacting protein, and possibly also motor proteins provide the scaffold for the formation of the protein complex as well as its intracellular localization.

Abstract-The activity of the endothelial nitric oxide synthase (eNOS) can be regulated independently of an increase in Ca 2ϩ by the phosphorylation of Ser 1177 but results only in a low nitric oxide (NO) output. In the present study, we assessed whether the agonist-induced (Ca 2ϩ -dependent, high-output) activation of eNOS is associated with changes in the phosphorylation of

Aging is considered to be the major risk factor for the development of atherosclerosis and, therefore, for coronary artery disease. Apart from age-associated remodeling of the vascular wall, which includes luminal enlargement, intimal and medial thickening, and increased vascular stiffness, endothelial function declines with age. This is most obvious from the attenuation of endothelium-dependent dilator responses, which is a consequence of the alteration in the expression and/or activity of the endothelial NO synthase, upregulation of the inducible NO synthase, and increased formation of reactive oxygen species. Aging is also associated with a reduction in the regenerative capacity of the endothelium and endothelial senescence, which is characterized by an increased rate of endothelial cell apoptosis.

Previous studies in the canine heart had shown that the growth of collateral arteries occurs via proliferative enlargement of pre-existing arteriolar connections (arteriogenesis). In the present study, we investigated the ultrastructure and molecular histology of growing and remodeling collateral arteries that develop after femoral artery occlusion in rabbits as a function of time from 2 h to 240 days after occlusion. Pre-existent arteriolar collaterals had a diameter of about 50 microm. They consisted of one to two layers of smooth muscle cells (SMCs) and were morphologically indistinguishable from normal arterioles. The stages of arteriogenesis consisted of arteriolar thinning, followed by transformation of SMCs from the contractile- into the proliferative- and synthetic phenotype. Endothelial cells (ECs) and SMCs proliferated, and SMCs migrated and formed a neo-intima. Intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) showed early upregulation in ECs, which was accompanied by accumulation of blood-derived macrophages. Mitosis of ECs and SMCs started about 24 h after occlusion, whereas adhesion molecule expression and monocyte adhesion occurred as early as 12 h after occlusion, suggesting a role of monocytes in vascular cell proliferation. Treatment of rabbits with the pro-inflammatory cytokine MCP-1 increased monocyte adhesion and accelerated vascular remodeling. In vitro shear-stress experiments in cultured ECs revealed an increased phosphorylation of the focal contacts after 30 min and induction of ICAM-1 and VCAM-1 expression between 2 h and 6 h after shear onset, suggesting that shear stress may be the initiating event. We conclude that the process of arteriogenesis, which leads to the positive remodeling of an arteriole into an artery up to 12 times its original size, can be modified by modulators of inflammation.

Background-Further to promoting angiogenesis, cell therapy may be an approach for cardiac regeneration. Recent

Endothelial cells situated at the interface between blood and the vessel wall play a crucial role in controlling vascular tone and homeostasis, particularly in determining the expression of pro- and anti-atherosclerotic genes. Many of these effects are mediated by changes in the generation and release of the vasodilator nitric oxide (NO) in response to hemodynamic stimuli exerted on the luminal surface of endothelial cells by the streaming blood (shear stress) and the cyclic strain of the vascular wall. The endothelial NO synthase (eNOS) is activated in response to fluid shear stress and numerous agonists via cellular events such as; increased intracellular Ca(2+), interaction with substrate and co-factors, as well as adaptor and regulatory proteins, protein phosphorylation, and through shuttling between distinct sub-cellular domains. Dysregulation of these processes leads to attenuated eNOS activity and reduced NO output which is a characteristic feature of numerous patho-physiological disorders such as diabetes and atherosclerosis. This review summarizes some of the recent findings relating to the molecular events regulating eNOS activity.

Abstract-TRPV4 is a broadly expressed Ca 2ϩ -permeable cation channel in the vanilloid subfamily of transient receptor potential channels. TRPV4 gates in response to a large variety of stimuli, including cell swelling, warm temperatures, the synthetic phorbol ester 4␣-phorbol 12,13-didecanoate (4␣-PDD), and the endogenous lipid arachidonic acid (AA). Activation by cell swelling and AA requires cytochrome P450 (CYP) epoxygenase activity to convert AA to epoxyeicosatrienoic acids (EETs) such as 5,6-EET, 8,9-EET, which both act as direct TRPV4 agonists. To evaluate the role of TRPV4 and its modulation by the CYP pathway in vascular endothelial cells, we performed Ca 2ϩ imaging and patch-clamp measurements on mouse aortic endothelial cells (MAECs) isolated from wild-type and TRPV4 Ϫ/Ϫ mice. All TRPV4-activating stimuli induced robust Ca 2ϩ responses in wild-type MAECs but not in MAECs isolated from TRPV4 Ϫ/Ϫ mice. Upregulation of CYP2C expression by preincubation with nifedipine enhanced the responses to AA and cell swelling in wild-type MAECs, whereas responses to other stimuli remained unaffected. Conversely, inhibition of CYP2C9 activity with sulfaphenazole abolished the responses to AA and hypotonic solution (HTS). Moreover, suppression of EET hydrolysis using 1-adamantyl-3-cyclo-hexylurea or indomethacin, inhibitors of soluble epoxide hydrolases (sEHs), and cyclooxygenases, respectively, enhanced the TRPV4-dependent responses to AA, HTS, and EETs but not those to 4␣-PDD or heat. Together, our data establish that CYP-derived EETs modulate the activity of TRPV4 channels in endothelial cells and shows the unraveling of novel modulatory pathways via CYP2C modulation and sEH inhibition. Key Words: TRP channels Ⅲ endothelium Ⅲ epoxygenases Ⅲ epoxide hydrolases T RPV4 is a Ca 2ϩ entry channel belonging to the vanilloid subfamily of the transient receptor potential (TRP) channels. 1,2 It is expressed in a broad range of tissues, including lung, spleen, kidney, testis, fat, brain, cochlea, skin, smooth muscle, liver, and vascular endothelium. [3][4][5] The physiological role of TRPV4 in these tissues may be highly divergent because TRPV4 is able to respond to a wide variety of physical, thermal, and chemical stimuli.Initially, TRPV4 was put forward as a mechanosensor or osmosensor. This was based on the finding that the channel opens in response to hypotonic cell swelling 3-8 as well as shear stress. 9 Indeed, mice lacking the TRPV4 gene show a disturbance of osmoregulation and an increased mechanical nociceptive threshold. 10,11 Furthermore, TRPV4 functions as a transducer of hypo-osmotic stimuli in primary afferent nociceptors 7 and plays an essential role in taxol-induced nociceptive behavioral responses to mechanical and hypotonic stimulation of the hind paw. 12 More recently, it was found that TRPV4 can also be activated by heating, 13,14 and that it is required for normal thermal responsiveness in vivo. The TRPV4 Ϫ/Ϫ mice "select" a warmer floor temperature in a gradient apparatus than their wild-type littermates ...