Endothelial Rictor is crucial for midgestational development and sustained and extensive FGF2-induced neovascularization in the adult.

To explore the general requirement of endothelial mTORC2 during embryonic and adolescent development, we knocked out the essential mTORC2 component Rictor in the mouse endothelium in the embryo, during adolescence and in endothelial cells in vitro. During embryonic development, Rictor knockout resulted in growth retardation and lethality around embryonic day 12. We detected reduced peripheral vascularization and delayed ossification of developing fingers, toes and vertebrae during this confined midgestational period. Rictor knockout did not affect viability, weight gain, and vascular development during further adolescence. However during this period, Rictor knockout prevented skin capillaries to gain larger and heterogeneously sized diameters and remodeling into tortuous vessels in response to FGF2. Rictor knockout strongly reduced extensive FGF2-induced neovascularization and prevented hemorrhage in FGF2-loaded matrigel plugs. Rictor knockout also disabled the formation of capillary-like networks by FGF2-stimulated mouse aortic endothelial cells in vitro. Low RICTOR expression was detected in quiescent, confluent mouse aortic endothelial cells, whereas high doses of FGF2 induced high RICTOR expression that was associated with strong mTORC2-specific protein kinase Cα and AKT phosphorylation. We demonstrate that the endothelial FGF-RICTOR axis is not required during endothelial quiescence, but crucial for midgestational development and sustained and extensive neovascularization in the adult.

Hypoxia potentiates tumor necrosis factor-α induced expression of inducible nitric oxide synthase and cyclooxygenase-2 in white and brown adipocytes.

Obesity involves hypoxic adipose tissue and low-grade chronic inflammation. We investigated the impact of hypoxia on inflammatory response to TNF-α in white and brown adipocytes. In response to TNF-α, the expression of the inducible enzymes iNOS and COX-2 was prominently and selectively potentiated during hypoxia while only moderately under normoxia. Levels of their products, nitrite and prostaglandinE2 were elevated accordingly. NS398, a selective COX-2 inhibitor, reduced nitrite levels. The expression of PGC-1α, a transcriptional co-activator involved in mitochondrial biogenesis, and PPARγ, a transcription factor involved in adipocyte homeostasis, was reduced by TNF-α during hypoxia. These results suggest that hypoxia potentiates the inflammatory response by TNF-α in both white and brown adipocytes and downregulates the transcription factors involved in adipocyte function.

Rictor in perivascular adipose tissue controls vascular function by regulating inflammatory molecule expression.

OBJECTIVE: Perivascular adipose tissue (PVAT) wraps blood vessels and modulates vasoreactivity by secretion of vasoactive molecules. Mammalian target of rapamycin complex 2 (mTORC2) has been shown to control inflammation and is expressed in adipose tissue. In this study, we investigated whether adipose-specific deletion of rictor and thereby inactivation of mTORC2 in PVAT may modulate vascular function by increasing inflammation in PVAT. APPROACH AND RESULTS: Rictor, an essential mTORC2 component, was deleted specifically in mouse adipose tissue (rictor(ad-/-)). Phosphorylation of mTORC2 downstream target Akt at Serine 473 was reduced in PVAT from rictor(ad-/-) mice but unaffected in aortic tissue. Ex vivo functional analysis of thoracic aortae revealed increased contractions and impaired dilation in rings with PVAT from rictor(ad-/-) mice. Adipose rictor knockout increased gene expression and protein release of interleukin-6, macrophage inflammatory protein-1α, and tumor necrosis factor-α in PVAT as shown by quantitative real-time polymerase chain reaction and Bioplex analysis for the cytokines in the conditioned media, respectively. Moreover, gene and protein expression of inducible nitric oxide synthase was upregulated without affecting macrophage infiltration in PVAT from rictor(ad-/-) mice. Inhibition of inducible nitric oxide synthase normalized vascular reactivity in aortic rings from rictor(ad-/-) mice with no effect in rictor(fl/fl) mice. Interestingly, in perivascular and epididymal adipose depots, high-fat diet feeding induced downregulation of rictor gene expression. CONCLUSIONS: Here, we identify mTORC2 as a critical regulator of PVAT-directed protection of normal vascular tone. Modulation of mTORC2 activity in adipose tissue may be a potential therapeutic approach for inflammation-related vascular damage.

Nuclear PIM1 confers resistance to rapamycin-impaired endothelial proliferation.

The PIM serine/threonine kinases and the mTOR/AKT pathway integrate growth factor signaling and promote cell proliferation and survival. They both share phosphorylation targets and have overlapping functions, which can partially substitute for each other. In cancer cells PIM kinases have been reported to produce resistance to mTOR inhibition by rapamycin. Tumor growth depends highly on blood vessel infiltration into the malignant tissue and therefore on endothelial cell proliferation. We therefore investigated how the PIM1 kinase modulates growth inhibitory effects of rapamycin in mouse aortic endothelial cells (MAEC). We found that proliferation of MAEC lacking Pim1 was significantly more sensitive to rapamycin inhibition, compared to wildtype cells. Inhibition of mTOR and AKT in normal MAEC resulted in significantly elevated PIM1 protein levels in the cytosol and in the nucleus. We observed that truncation of the C-terminal part of Pim1 beyond Ser 276 resulted in almost exclusive nuclear localization of the protein. Re-expression of this Pim1 deletion mutant significantly increased the proliferation of Pim1(-/-) cells when compared to expression of the wildtype Pim1 cDNA. Finally, overexpression of the nuclear localization mutant and the wildtype Pim1 resulted in complete resistance to growth inhibition by rapamycin. Thus, mTOR inhibition-induced nuclear accumulation of PIM1 or expression of a nuclear C-terminal PIM1 truncation mutant is sufficient to increase endothelial cell proliferation, suggesting that nuclear localization of PIM1 is important for resistance of MAEC to rapamycin-mediated inhibition of proliferation.

B2-kinin receptor plays a key role in B1-, angiotensin converting enzyme inhibitor-, and vascular endothelial growth factor-stimulated in vitro angiogenesis in the hypoxic mouse heart.

AIMS: Angiotensin converting enzyme (ACE) inhibition reduces heart disease and vascular stiffness in hypertension and leads to kinin accumulation. In this study, we analysed the role and importance of two kinin receptor subtypes in angiogenesis during ACE inhibition in an in vitro model of angiogenesis of the mouse heart. METHODS AND RESULTS: First, we analysed the angiogenic properties of bradykinin and enalapril on wild-type C57Bl/6 and B2 receptor(-/-) mouse heart under normoxia (21% O(2)) and hypoxia (1% O(2)) in vitro and the contribution of B1 and B2 kinin receptors to this effect. Bradykinin induced dose-dependent endothelial sprout formation in vitro in adult mouse heart only under hypoxia (1.7 fold, n = 6, P < 0.05). The B2 receptor mediated sprouting that was induced by bradykinin and vascular endothelial growth factor (VEGF(164); n = 6, P < 0.05), but did not mediate sprouting that was induced by growth factors bFGF or PDGF-BB. Enalapril induced sprouting through both the B1 and B2 kinin receptors, but it required the presence of the B2 receptor in both scenarios and was dependent on BK synthesis. B1-receptor agonists induced sprout formation via the B1 receptor (2.5 fold, n = 6, P < 0.05), but it required the presence of the B2 receptor for them to do so. Both B2-receptor and B1-receptor agonist-induced angiogenesis required nitric oxide biosynthesis. CONCLUSION: 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.

Formation of new blood vessels in the heart can be studied in cell cultures.

Controlled induction of the formation of new microvessels, i.e., therapeutic angiogenesis, may be used one day to treat patients that for example had suffered a myocardial infarction. Experimental models of angiogenesis in the heart in vivo substantially stress the animal. We therefore developed a model of angiogenesis of the heart in vitro, where mouse and rat heart pieces are stimulated under controlled conditions in a three dimensional matrix. Capillary-like sprouts emerging in these cultures represent early to midterm steps of angiogenesis and can be quantified to study potential angiogenic compounds and underlying mechanisms.

Hypoxia-induced endothelial proliferation requires both mTORC1 and mTORC2.

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 (>30 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 (P<0.05; n> or =4). Similarly, hypoxia induced Akt-dependent and rapamycin-sensitive proliferation in mouse embryonic fibroblasts. This response was partially blunted by hypoxia-inducible factor-1alpha knockdown and not affected by TSC2 knockout. Finally, mTORC2 inhibition by rictor silencing, especially (P<0.001; n=7), and mTORC1 inhibition by raptor silencing, partially (P<0.05; n=7), 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.

Hypoxia enhances vascular cell proliferation and angiogenesis in vitro via rapamycin (mTOR)-dependent signaling.

Angiogenesis and vascular cell proliferation are pivotal in physiological and pathological processes including atherogenesis, restenosis, wound healing, and cancer development. Here we show that mammalian target of rapamycin (mTOR) signaling plays a key role in hypoxia-triggered smooth muscle and endothelial proliferation and angiogenesis in vitro. Hypoxia significantly increased DNA synthesis and proliferative responses to platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) in rat and human smooth muscle and endothelial cells. In an in vitro 3-dimensional model of angiogenesis, hypoxia increased PDGF- and FGF-stimulated sprout formation from rat and mouse aortas. Hypoxia did not modulate PDGF receptor mRNA, protein, or phosphorylation. PI3K activity was essential for cell proliferation under normoxic and hypoxic conditions. Activities of PI3K-downstream target PKB under hypoxia and normoxia were comparable. However, mTOR inhibition by rapamycin specifically abrogated hypoxia-mediated amplification of proliferation and angiogenesis, but was without effect on proliferation under normoxia. Accordingly, hypoxia-mediated amplification of proliferation was further augmented in mTOR-overexpressing endothelial cells. Thus, signaling via mTOR may represent a novel mechanism whereby hypoxia augments mitogen-stimulated vascular cell proliferation and angiogenesis.