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 serinethreonine 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 antiapoptotic pathways mediated by Akt and survivin in endothelial cells may contribute to Ang-1 stabilization of vascular structures during angiogenesis, in vivo.During angiogenesis, endothelial cells receive cues from growth factors to initiate mitosis, migration, and organization of endothelial cells into primitive angiotubes and patent vascular networks (1, 2). These processes critically depend on preservation of endothelial cell viability. Disruption of endothelial cell-matrix contacts or interference with extracellular survival signals is sufficient to initiate caspase-dependent apoptosis in endothelium, culminating with rapid involution of vascular structures (3, 4). Unlike most angiogenic regulators, including fibroblast growth factor or vascular endothelial growth factor (VEGF), 1 angiopoietin-1 (Ang-1) does not stimulate endothelial cell growth but rather promotes stabilization of vascular networks and branching morphogenesis in vivo and in vitro (5-8). Little is known about the signaling requirements of these responses, and the mechanism(s) of Ang-1-induced cytoprotection are unknown (7, 9). The major goal of this paper was to elucidate a potential link between endothelial cell viability and maintenance of angiogenesis by examining the ability of Ang-1 to activate the antiapoptotic serine-threonine kinase, Akt (or protein kinase B). Moreover, we examined the relationship between Ang-1, Akt activation, and the expression of the anti-apoptotic genes, bcl-2 and survivin, in cultured microvascular endothelial cells (MVEC). MATERIALS AND METHODSCell Culture and Reagents-Bovine MVEC (Vec Technologies, Rensselaer, NY) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, L-glutamine, and antibiotics (penicillin and streptomycin). Cells (up to passage 12) were used for the experiments. In experiments examining endogenous survivin expression, human umbilical vein endothelial cells (HUVEC) were used, because the survivin antibody recognized human survivin better than bovine survivin. HUVEC were cultured on gelatin-coated tissue culture flasks in M199 medium containing 20% fetal bovine serum, 50 g/ml endothelial cell growth supplement (a commercial preparation that contains main...

Recent efforts to identify treatments for myocardial ischemia reperfusion injury have resulted in the discovery of a novel series of highly potent α,α-disubstituted amino acid-based arginase inhibitors. The lead candidate, (R)-2-amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic acid, compound 9, inhibits human arginases I and II with IC50s of 223 and 509 nM, respectively, and is active in a recombinant cellular assay overexpressing human arginase I (CHO cells). It is 28% orally bioavailable and significantly reduces the infarct size in a rat model of myocardial ischemia/reperfusion injury. Herein, we report the design, synthesis, and structure-activity relationships (SAR) for this novel series of inhibitors along with pharmacokinetic and in vivo efficacy data for compound 9 and X-ray crystallography data for selected lead compounds cocrystallized with arginases I and II.

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 [LM) and cyclo-oxygenase with indomethacin (2.8 AM) 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 pM) and 7-ethoxyresorufin (1 JLM). 17-Octadecynoic acid (17-ODYA 2 pM), 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.

SUMMARY: 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 dosedependent 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. (Lab Invest 2001, 81:327-334).

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Abstract-Tumor necrosis factor-␣ (TNF-␣) 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-␣ or Ang II; mRNA accumulation peaked at 2 hours and decreased to control levels by Ϸ6 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-␣ or Ang II. Both the increase in COX-2 mRNA accumulation and protein expression in response to either TNF-␣ 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-␣-and Ang II-mediated increases in DNA content and cell number by Ϸ95%. 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-␣ or Ang II. (Circ Res. 2000;86:906-914.)

This article highlights our work toward the identification of a potent, selective, and efficacious acidic mammalian chitinase (AMCase) inhibitor. Rational design, guided by X-ray analysis of several inhibitors bound to human chitotriosidase (hCHIT1), led to the identification of compound 7f as a highly potent AMCase inhibitor (IC values of 14 and 19 nM against human and mouse enzyme, respectively) and selective (>150× against mCHIT1) with very good PK properties. This compound dosed once daily at 30 mg/kg po showed significant anti-inflammatory efficacy in HDM-induced allergic airway inflammation in mice, reducing inflammatory cell influx in the BALF and total IgE concentration in plasma, which correlated with decrease of chitinolytic activity. Therapeutic efficacy of compound 7f in the clinically relevant aeroallergen-induced acute asthma model in mice provides a rationale for developing AMCase inhibitor for the treatment of asthma.

A defining characteristic of pulmonary hypertension (PH) is the extensive remodeling of pulmonary arteries (PAs), which results in progressive increases in vascular resistance and stiffness and eventual failure of the right ventricle. There is no cure for PH and identification of novel molecular mechanisms that underlie increased proliferation, reduced apoptosis, and excessive extracellular matrix production in pulmonary artery smooth muscle cells (PASMCs) is a vital objective. Galectin-3 (Gal-3) is a chimeric lectin and potent driver of many aspects of fibrosis, but its role in regulating PASMC behavior in PH remains poorly understood. Herein, we evaluated the importance of increased Gal-3 expression and signaling on PA vascular remodeling and cardiopulmonary function in experimental models of PH. Gal-3 expression was quantified by qRT-PCR, immunoblotting, and immunofluorescence imaging, and its functional role was assessed by specific Gal-3 inhibitors and CRISPR/Cas9-mediated knockout of Gal-3 in the rat. In rat models of PH, we observed increased Gal-3 expression in PASMCs, which stimulated migration and resistance to apoptosis, whereas silencing or genetic deletion reduced cellular migration and PA fibrosis and increased apoptosis. Gal-3 inhibitors attenuated and reversed PA remodeling and fibrosis, as well as hemodynamic indices in monocrotaline (MCT)-treated rats in vivo. These results were supported by genetic deletion of Gal-3 in both MCT and Sugen Hypoxia rat models. In conclusion, our results suggest that elevated Gal-3 levels contribute to inappropriate PA remodeling in PH by enhancing multiple profibrotic mechanisms. Therapeutic strategies targeting Gal-3 may be of benefit in the treatment of PH.