Nicholas G. Theodorakis

The role of nitric oxide synthase isoforms in extrahepatic portal hypertension: studies in gene-knockout mice

BACKGROUND & AIMS Considerable debate exists concerning which isoform of nitric oxide synthase (NOS) is responsible for the increased production of NO in PHT. We used the portal vein ligation model of PHT in wild-type and eNOS- or iNOS-knockout mice to definitively determine the contribution of these isoforms in the development of PHT. METHODS The portal vein of wild-type mice, or those with targeted mutations in the nos2 gene (iNOS) or the nos3 gene (eNOS), was ligated and portal venous pressure (Ppv), abdominal aortic blood flow (Qao), and portosystemic shunt determined 2 weeks later. RESULTS In wild-type mice, as compared with sham-operated controls, portal vein ligation (PVL) resulted in a time-dependent increase in Ppv (7.72 +/- 0.37 vs 17.57 +/- 0.51 cmH(2)O, at 14 days) concomitant with a significant increase in Qao (0.12 +/- 0.003 vs 0.227 +/- 0.005 mL/min/g) and portosystemic shunt (0.47% +/- 0.01% vs 84.13% +/- 0.09% shunt). Likewise, PVL in iNOS-deficient mice resulted in similar increases in Ppv, Qao, and shunt development. In contrast, after PVL in eNOS-deficient animals, there was no significant change in Ppv (7.52 +/- 0.22 vs 8.07 +/- 0.4 cmH(2)0) or Qao (0.111 +/- 0.01 vs 0.14 +/-.023 mL/min/g). However, eNOS (-/-) mice did develop a substantial portosystemic shunt (0.33% +/- 0.005% vs 84.53% +/- 0.19% shunt), comparable to that seen in wild-type animals after PVL. CONCLUSIONS These data support a key role for eNOS, rather than iNOS, in the pathogenesis of PHT.

Pulsatile Flow–Induced Angiogenesis

Objective—Angiogenesis plays a key role in the growth and function of normal and pathological tissues. We investigated the effect of pulsatile flow on endothelial cell (EC) in vitro angiogenic activity. Methods and Results—Bovine aortic ECs were exposed to “static” or “flow” (1.2 to 67.0 mL/min, shear stress 1.4 to 19.2 dyne/cm2) conditions for 2 to 24 hours. After exposure, angiogenesis was measured as tubule formation on Matrigel, and EC migration was assessed by filter migration assay. Pulsatile flow increased angiogenesis and EC migration in a temporal and force-dependent manner, with a maximal effect at 16 hours (13.2 dyne/cm2). Pertussis toxin completely inhibited the effect of pulsatile flow on angiogenesis and migration. Transfection of ECs with inhibitory mutants of the &agr; subunit of Gi1 or Gi3, but not Gi2, inhibited the flow-induced angiogenic response by 61±2% and 32±6%, respectively, whereas transfection with constitutively activated mutants of the &agr; subunit of Gi1 or Gi3, but not Gi2, increased the flow-induced response by 202±23% and 70±4%, respectively. In contrast, inhibition of G&bgr;&ggr; by the carboxy terminal fragment of &bgr;-adrenergic receptor kinase overexpression increased the flow-induced response by 82±8%. Conclusions—These results suggest that pulsatile flow stimulates angiogenesis and that this effect is mediated by activation of Gi&agr;1 or Gi&agr;3, but not G&bgr;&ggr;, subunits.

Role of cyclic-AMP responsive element binding (CREB) proteins in cell proliferation in a rat model of hepatocellular carcinoma

The role of cyclic adenosine monophosphate (cAMP) is poorly understood in the regulation of normal and abnormal hepatic cell growth. In this study, we examined the regulation of intracellular cAMP levels and its effect on nuclear cAMP responsive elements (CREs) in a rat model of hepatocellular carcinoma (HCC). Tumorigenic liver cells were cultured from an in vivo model of HCC and the role of cAMP in cell mitogenesis determined. These data demonstrated agents that elevate intracellular cAMP ([cAMP]i) levels caused significant dose‐dependent inhibition of serum‐stimulated mitogenesis in HCC cells. Cells were next analyzed for transcription factor expression and activity following increased [cAMP]i. These data demonstrated time‐ and dose‐dependent increases in CRE binding protein (pCREB) activity, a maximal response occurring after 10–20 min before returning to basal levels within 60 min. In contrast, increased [cAMP]i levels led to sustained inducible cAMP early repressor (ICER) II/IIγ mRNA and protein induction. To understand these data in relation to the in vivo setting, HCC tumors were analyzed and compared to pair‐matched normal liver (NL) samples. These studies demonstrated significantly elevated Gsα‐protein expression in HCC versus NL in the absence of significant changes in basal cAMP levels. Analysis of total and active CREB demonstrated significantly increased total CREB/pCREB in HCC versus NL. Further analysis of CRE expression demonstrated significantly increased expression of ICER mRNA and protein in HCC versus sham operated (Sh). These data demonstrate cAMP, while capable of stimulating promitogenic CREB activation inhibits cell mitogenesis in HCC possibly via ICER induction. J. Cell. Physiol. 206: 411–419, 2006.

Ethanol inhibits pulse pressure-induced vascular smooth muscle cell migration by differentially modulating plasminogen activator inhibitor type 1, matrix metalloproteinase-2 and -9

We investigated the effect of ethanol on the pulse pressure-induced expression of PAI-1 and MMP-2/9 in human smooth muscle cells (SMC). Human SMC were exposed to static or pulse pressure (25 mL/min; pulse pressure 106/50 mm Hg) conditions for 24 h in the absence or presence of ethanol (0.1-100 mM). SMC migration was then measured by Transwell migration assay. SMC exposed to pulse pressure demonstrated a significant increase in PAI-1 mRNA and protein expression (approximately 4-fold and approximately 3-fold) concomitant with a 3- and 8-fold increase in MMP-2 and MMP-9 protein, respectively. Ethanol dose-dependently inhibited the pulse pressure-induced SMC migration with complete inhibition observed at 20 mM. There was no effect of ethanol on basal PAI-1 or MMP-2/9 in SMC under static conditions. However, ethanol significantly enhanced the pulse pressure-induced PAI-1 mRNA and protein expression (2.2 +/- 0.52 fold and 2.5 +/- 0.27 fold, for 10 mM), respectively. In contrast, ethanol dose-dependently inhibited the pulse pressure-induced increases in MMP-9 protein and pro-MMP-9 activity and to a lesser extent MMP-2 mRNA and protein and pro-MMP-2 activity, with significant inhibition observed at 1 mM. These data provide a molecular mechanism mediating the inhibitory effect of ethanol on pulse-pressure-induced SMC migration and may be relevant to the cardioprotective effects of ethanol in vivo.