Interleukin-11 up-regulates survivin expression in endothelial cells through a signal transducer and activator of transcription-3 pathway.

Interleukin-11 (IL-11) reduces injury both in vivo and in vitro, but the mechanisms are unknown. Stimulation of serum- and growth factor-deprived HUVEC with IL-11 increased survivin mRNA and protein expression levels in a dose-dependent manner, with maximal induction at 50 to 100 ng/ml of IL-11. Survivin mRNA expression peaked after 3 to 6 hours of IL-11 treatment and decreased by 24 hours. Survivin protein expression was maximal at 6 hours of treatment and remained elevated through 24 hours. Survivin induction may be mediated by activation of protein kinase B/Akt, but IL-11 failed to activate this pathway in HUVEC. IL-11 did activate signal transducer and activator of transcription (STAT)-3 and IL-11 failed to induce survivin expression in HUVEC transduced with a dominant-negative STAT3 mutant, whereas control-transduced HUVEC responded normally. An IL-11 transgene caused increased survivin mRNA expression in mice compared with control littermates. Intradermal injection of IL-11 (500 ng) into human skin xenografts on immunodeficient mice up-regulated survivin protein in microvascular endothelium and epithelial keratinocytes. We conclude that IL-11 induces expression of survivin, an antiapoptotic protein, in vitro and in vivo, and identify STAT3 as a critical mediator of this response.

Cyclooxygenase-2 is required for tumor necrosis factor-alpha- and angiotensin II-mediated proliferation of vascular smooth muscle cells.

Tumor necrosis factor-alpha (TNF-alpha) and angiotensin II (Ang II) induced a transient increase in vascular smooth muscle cell (VSMC) cyclooxygenase-2 (COX-2) mRNA accumulation, without affecting COX-1 mRNA levels. The kinetics of COX-2 mRNA accumulation were similar in VSMCs challenged with either TNF-alpha or Ang II; mRNA accumulation peaked at 2 hours and decreased to control levels by approximately 6 hours. Accumulation of COX-2 mRNA was associated with a time-dependent increase of COX-2 protein expression that displayed similar kinetics in response to either TNF-alpha or Ang II. Both the increase in COX-2 mRNA accumulation and protein expression in response to either TNF-alpha or Ang II were inhibited by the mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor PD098059. In addition, the AT(1)-selective receptor antagonist losartan attenuated the Ang II-mediated increase in COX-2 mRNA accumulation; the AT(2)-selective antagonist PD123319 had no effect. Prostacyclin I(2) synthesis was tightly coupled to expression of COX-2, whereas prostaglandin E(2) and thromboxane A(2) (TXA(2)) synthesis may be associated with differential usage of COX-1 and COX-2. The COX-2-selective inhibitors NS-398 and nimesulide and the TXA(2) receptor antagonist BMS 180,291 inhibited TNF-alpha- and Ang II-mediated increases in DNA content and cell number by approximately 95%. These findings suggest that a prostanoid derived from COX-2, possibly TXA(2), may contribute to VSMC hyperplasia in vessel injury or pathophysiological conditions associated with elevated levels of either TNF-alpha or Ang II.

Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway.

A productive angiogenic response must couple to the survival machinery of endothelial cells to preserve the integrity of newly formed vessels. Angiopoietin-1 (Ang-1) is an endothelium-specific ligand essential for embryonic vascular stabilization, branching morphogenesis, and post-natal angiogenesis, but its contribution to endothelial cell survival has not been completely elucidated. Here we show that Ang-1 acting via the Tie 2 receptor induces phosphorylation of the survival serine-threonine kinase, Akt (or protein kinase B). This is associated with up-regulation of the apoptosis inhibitor, survivin, in endothelial cells and protection of endothelium from death-inducing stimuli. Moreover, dominant negative survivin negates the ability of Ang-1 to protect cells from undergoing apoptosis. The activation of anti-apoptotic pathways mediated by Akt and survivin in endothelial cells may contribute to Ang-1 stabilization of vascular structures during angiogenesis, in vivo.

IL-11 activates human endothelial cells to resist immune-mediated injury.

IL-11, a gp130-signaling cytokine, is protective in several in vivo models of immune-mediated and inflammatory injury. HUVECs express IL-11 receptor alpha-chain and gp130. Human IL-11 causes rapid (2-10 min) tyrosine phosphorylation of gp130. IL-11 at 0.1 and 10 ng/ml induces tyrosine phosphorylation of STAT3 and STAT1, respectively, although maximal responses require 50 ng/ml. Phospho-STAT3 and phospho-STAT1 levels peak rapidly (2.5 min) and disappear by 60 min. The p42 and p44 mitogen-activated protein kinases (MAPKs) are phosphorylated in response to 0.3 ng/ml IL-11 with maximal activation at 30 ng/ml IL-11. Phosphorylation of p42 and p44 MAPKs, which can be prevented by a mitogen-activated protein/extracellular signal-related kinase kinase-1 inhibitor, peaks by 15-20 min and largely disappears by 40 min. IL-11 does not activate NF-kappaB nor does it inhibit NF-kappaB activation by TNF. Similarly, IL-11 neither induces E-selectin or ICAM-1 nor blocks induction by TNF. Although IL-11 does not alter class I MHC complex molecule expression, pretreatment with 0.5 ng/ml IL-11 partially protects HUVECs against lysis by allospecific class I MHC-restricted cytolytic T lymphocytes or by anti-class I MHC Ab plus heterologous C. IL-11-induced cytoprotection is protein synthesis dependent and may depend on mitogen-activated protein/extracellular signal-related kinase kinase-1. Our results indicate that low (i.e., STAT3- and MAPK-activating) concentrations of IL-11 confer resistance to immune-mediated injury in cultured HUVECs without inhibiting proinflammatory responses.

Tyrosine phosphatase-dependent/tyrosine kinase-independent induction of nuclear factor-kappa B by tumor necrosis factor-alpha: effects on prostaglandin endoperoxide synthase-2 mRNA accumulation.

We previously have demonstrated that tumor necrosis factor-alpha (TNF-alpha) increases prostaglandin endoperoxide synthase-2 (PGHS-2) mRNA accumulation and tyrosine phosphorylation in the fibrosarcoma cell line, MCA-101. Tyrosine kinase inhibitor, genistein, and tyrosine phosphatase inhibitor, phenylarsine oxide (PAO), blocked TNF-alpha-mediated induction of PGHS-2 mRNA in these cells. Because the PGHS-2 promoter has a nuclear factor-kappa B (NF-kappa B) binding motif, which is important for PGHS-2 gene transcription in some cell types, we have evaluated the effects of tyrosine kinase inhibitors and PAO on TNF-alpha-induced NF-kappa B activation. TNF-alpha (1 nM) rapidly induced translocation of NF-kappa B, an event accompanied by degradation of inhibitory protein I kappa B-alpha. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a serine protease inhibitor, inhibited I kappa B-alpha degradation and NF-kappa B activation in response to TNF-alpha in a dose-dependent manner (25, 50, 100 microM). TPCK also inhibited PGHS-2 mRNA accumulation. These data suggest that NF-kappa B contributed to PGHS-2 mRNA accumulation in MCA-101 cells stimulated with TNF-alpha. PAO (2.4 microM) completely abolished activation of NF-kappa B and degradation of I kappa B-alpha induced by TNF-alpha at a concentration that blocked PGHS-2 mRNA accumulation. However, four tyrosine kinase inhibitors, genistein, tyrphostin 47, herbimycin A and erbstatin, failed to block translocation of NF-kappa B and degradation of I kappa B-alpha. These data demonstrate that tyrosine kinase pathways are not required for TNF-alpha-induced NF-kappa B activation in MCA-101 cells and suggest that signaling via these pathways mediates TNF-alpha-induced PGHS-2 mRNA accumulation via an NF-kappa B-independent mechanism. Moreover, an upstream tyrosine phosphatase pathway may mediate PGHS-2 mRNA accumulation by TNF-alpha via an NF-kappa B-dependent mechanism.

Tumour necrosis factor-alpha-dependent regulation of prostaglandin endoperoxide synthase-2.

Tumour necrosis factor alpha (TNF-alpha)-mediated regulation of prostaglandin endoperoxide synthase-2 (PGHS-2) mRNA levels was examined in murine fibrosarcoma MCA-101 cells. We demonstrated that the formation of prostaglandin E2 (PGE2) is highly dependent on the expression of PGHS-2 enzyme in these cells. TNF-alpha-induced PGE2 production was evident after 12 h and was associated with a significant TNF-alpha-mediated increase in PGHS-2 immunoreactive protein. A specific PGHS-2 inhibitor, NS-398, completely abolished the TNF-alpha-mediated increase in PGE2 production, suggesting that the PGE2 formed in response to TNF-alpha was derived from PGHS-2. TNF-alpha-mediated PGHS-2 mRNA accumulation was observed at 1 h, remained elevated for 24 h, and was blocked by actinomycin D, indicating that TNF-alpha increases PGHS-2 gene transcription. A significant post-transcriptional mechanism also contributed to the increased PGHS-2 mRNA accumulation as the mRNA half-life was approximately 4-5 h in TNF-alpha-stimulated cells. Inhibition of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) inhibited the TNF-alpha-mediated increase in PGHS-2 mRNA levels. We suggest that PTPs and PTKs play a role in the transcriptional and/or post-transcriptional mechanisms that contribute to the regulation of the PGHS-2 gene by TNF-alpha.

Induction of prostaglandin endoperoxide synthase-2 by serine-threonine phosphatase inhibition.

Regulation of prostaglandin endoperoxide synthase-2 (PGHS-2) mRNA levels by serine-threonine phosphatases was examined in murine fibrosarcoma methylcholanthrene-101 cells. Okadaic acid (OA), a serine-threonine phosphatase inhibitor, induced PGE2 production and a significant increase in PGHS-2 immunoreactive protein. A specific PGHS-2 inhibitor, N-(2-cyclohexyloxy-4-nitrophenyl) methanesulphonamide, completely abolished the OA-mediated increase in PGE2 production, which suggests that the PGE2 formed in response to OA was derived from PGHS-2. OA-mediated PGHS-2 mRNA accumulation was observed at 1 hr, remained elevated for 24 hr and was blocked by actinomycin D, which indicates that OA increases PGHS-2 gene transcription. A significant post-transcriptional mechanism also contributed to the increased PGHS-2 mRNA accumulation, because the mRNA half-life was approximately 4 to 5 h in OA-stimulated cells. Tumor necrosis factor-alpha, but not OA, activated transcription factor nuclear factor-kappaB in methylcholanthrene-101 cells, as demonstrated by translocation of the nuclear factor-kappaB complex to the nucleus and disappearance of the cytoplasmic inhibitory protein, IkappaB-alpha. We conclude that inhibition of serine-threonine phosphatases contributes to the up-regulation of PGHS-2 expression in an NF-kappaB-independent manner.

Cytochrome P450-dependent effects of bradykinin in the rat heart.

1. Vasodilator responses to bradykinin (BK) in the rat heart are reported to be independent of NO and cyclo-oxygenase/lipoxygenase products of arachidonic acid (AA). 2. We verified that inhibition of NO synthase with L-nitroarginine (50 microM) and cyclo-oxygenase with indomethacin (2.8 microM) were without effect on vasodilator responses to BK (10-1000 ng) in the Langendorff rat heart preparation. 3. L-Nitroarginine elevated perfusion pressure, signifying a crucial role of NO in the maintenance of basal vasculature tone. 4. In hearts treated with L-nitroarginine to eliminate NO and elevate perfusion pressure, vasodilator responses were reduced by inhibitors of cytochrome P450 (P450), clotrimazole (1 microM) and 7-ethoxyresorufin (1 microM). 17-Octadecynoic acid (17-ODYA 2 microM), a mechanism based inhibitor of P450-dependent metabolism of fatty acids, also reduced vasodilator responses to BK. 5. These results confirm that NO and prostaglandins do not mediate vasodilator responses to BK in the rat heart but suggest a major role for a P450-dependent mechanism via AA metabolism.

The antiproteinuric action of enalapril in stroke-prone spontaneously hypertensive rats is unrelated to alterations in urinary prostaglandins.

We examined whether the renal protective effect of the angiotensin I converting enzyme inhibitor enalapril in stroke-prone spontaneously hypertensive rats (SHRSP) is dose-related and associated with alterations in the urinary excretion of prostaglandin (PG) E2 and 6-keto-PGF1 alpha, a stable breakdown product of prostacyclin. Enalapril maleate at 1.5, 5 and 15 mg/kg/day or vehicle was chronically administered to saline-drinking SHRSP (six per group) starting at 8.1 weeks of age. Vehicle-treated SHRSP developed severe hypertension, proteinuria and strokes (age at death, 14 +/- 1 weeks; mean +/- S.E.). Enalapril prolonged survival dose-dependently and reduced proteinuria; all SHRSP given 15 mg/kg/day lived beyond 23 weeks of age without evidence of stroke or proteinuria. There was no effect of enalapril at any dose on systolic arterial blood pressure in spite of variable levels of urinary protein excretion and onset of stroke in the different groups. Likewise, urinary 6-keto-PGF1 alpha and PGE2 excretion did not differ among the groups except for an increase in 6-keto-PGF1 alpha in the 15 mg/kg/day group at one week after initiation of enalapril therapy. These results are consistent with a dose-related renal protective action of enalapril in saline-drinking SHRSP that is not closely associated with sustained alterations in urinary excretion of renal vasodilatory PGs.