Abstract-A central regulator of cell growth that has been implicated in responses to stress such as hypoxia is mTOR (mammalian Target Of Rapamycin). We have shown previously that mTOR is required for angiogenesis in vitro and endothelial cell proliferation in response to hypoxia. Here we have investigated mTOR-associated signaling components under hypoxia and their effects on cell proliferation in rat aortic endothelial cells (RAECs). Hypoxia (1% O 2 ) rapidly (Ͼ30 minutes) and in a concentration-dependent manner promoted rapamycin-sensitive and sustained phosphorylation of mTOR-Ser2448 followed by nuclear translocation in RAECs. Similarly, hypoxia induced phosphorylation of the mTORC2 substrate Akt-Ser473 (3 to 6 hours at 1% O 2 ) and a brief phosphorylation peak of the mTORC1 substrate S6 kinase-Thr389 (10 to 60 minutes). Phosphorylation of Akt was inhibited by mTOR knockdown and partially with rapamycin. mTOR knockdown, rapamycin, or Akt inhibition specifically and significantly inhibited proliferation of serum-starved RAECs under hypoxia (PϽ0.05; nՆ4). Similarly, hypoxia induced Akt-dependent and rapamycinsensitive proliferation in mouse embryonic fibroblasts. This response was partially blunted by hypoxia-inducible factor-1␣ knockdown and not affected by TSC2 knockout. Finally, mTORC2 inhibition by rictor silencing, especially (PϽ0.001; nϭ7), and mTORC1 inhibition by raptor silencing, partially (PϽ0.05; nϭ7), inhibited hypoxia-induced RAEC proliferation. Thus, mTOR mediates an early response to hypoxia via mTORC1 followed by mTORC2, promoting endothelial proliferation mainly via Akt signaling. mTORC1 and especially mTORC2 might therefore play important roles in diseases associated with hypoxia and altered angiogenesis. (Circ Res. 2007;100:79-87.)

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Abstract-Angiotensin II is a vasoactive peptide that may affect vascularization of the ischemic heart via angiogenesis. In this study we aimed at studying the mechanisms underlying the angiogenic effects of angiotensin II under hypoxia in the mouse heart in vitro. Endothelial sprout formation from pieces of mouse hearts was assessed under normoxia (21% O 2 ) and hypoxia (1% O 2 ) during a 7-day period of in vitro culture. Only under hypoxia did angiotensin II dose-dependently induce endothelial sprout formation, peaking at 10 Ϫ7 mol/L of angiotensin II. Angiotensin II type 1 (AT 1 ) receptor blockade by losartan did not affect angiotensin II-induced sprouting in wild-type mice. Conversely, the angiotensin II type 2 (AT 2 ) receptor antagonist PD 123319 blocked this response. In hearts from AT 1 Ϫ/Ϫ mice, angiotensin II-elicited sprouting was preserved but blocked again by AT 2 receptor antagonism. In contrast, no angiotensin II-induced sprouting was found in preparations from hearts of AT 2 Ϫ/Ϫ mice. Angiotensin II-mediated angiogenesis was also abolished by a specific inhibitor of the B2 kinin receptor in both wild-type and AT 1 Ϫ/Ϫ mice. Furthermore, angiotensin II failed to induce endothelial sprout formation in hearts from B2 Ϫ/Ϫ mice. Finally, NO inhibition completely blunted sprouting in hearts from wild-type mice, whereas NO donors could restore sprouting in AT 2 Ϫ/Ϫ and B2 Ϫ/Ϫ hearts. This in vitro study suggests the obligatory role of hypoxia in the angiogenic effect of angiotensin II in the mouse heart via the AT 2 receptor through a mechanism that involves bradykinin, its B2 receptor, and NO as a downstream effector. Key Words: heart Ⅲ angiotensin II Ⅲ bradykinin Ⅲ losartan Ⅲ nitric oxide I schemic heart disease and left ventricular hypertrophy are characterized by impaired cardiac function caused by, among others, inadequate blood supply to the myocardium. In order to relieve this condition, blood flow to the myocardium needs to be restored by remodeling of pre-existing unused collateral blood vessels (arteriogenesis) and by the growth of new microvessels (angiogenesis). This process may also prevent the death and promote regeneration of damaged myocardial tissue.Angiogenic stimuli are generated by hypoxia through activation of endothelial cell signaling 1 and gene transcription of key angiogenic molecules, such as vascular endothelial growth factor (VEGF). 2 In mice, activation of pre-existing collateral vascularization that restores blood flow to the acutely ischemic heart was shown to be induced by angiotensin II (Ang II), 3 a key regulator of blood pressure and the main effector of the reninangiotensin-aldosterone system. 4 During ischemia or cancer, Ang II was shown to induce angiogenesis. 5 Two major subtypes of Ang II receptors are expressed in the myocardium, 6 Ang II type 1 (AT 1 ) and Ang II type 2 (AT 2 ) receptors. 7,8 Most of the Ang II cardiovascular effects, for example, vasoconstriction, are attributed to AT 1 . 9 AT 1 is an ubiquitous receptor that presents 2 subtypes in rodents of a...

The kinin B2 receptor plays a crucial role in angiogenesis that is induced by different vasoactive molecules, namely bradykinin, ACE inhibitors, B1-stimulating kinin metabolites, and VEGF164 in an in vitro model of angiogenesis of mouse heart under hypoxia. Therapeutic treatment of hypertensive patients by using ACE inhibitors may potentially benefit the ischaemic heart through inducing B2-dependent heart neovascularization.

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