Nicholas A. Flavahan

Norepinephrine Up-regulates the Expression of Vascular Endothelial Growth Factor, Matrix Metalloproteinase (MMP)-2, and MMP-9 in Nasopharyngeal Carcinoma Tumor Cells

Recent studies using ovarian cancer cells have shown that the catecholamine hormones norepinephrine (norepi) and epinephrine (epi) may influence cancer progression by modulating the expression of matrix metalloproteinases (MMP) and vascular endothelial growth factor (VEGF). The purpose of this study is to determine if the stress hormone norepi can influence the expression of MMP-2, MMP-9, and VEGF in nasopharyngeal carcinoma (NPC) tumors by using three NPC tumor cell lines. The NPC cell lines HONE-1, HNE-1, and CNE-1 were treated with norepi. The effects of norepi on MMP-2, MMP-9, and VEGF synthesis were measured by ELISA; functional MMP activity was measured by the invasive potential of the cells using a membrane invasion culture system whereas functional activity of VEGF was analyzed using a human umbilical vein endothelial cell tube formation assay. Norepi treatment increased MMP-2, MMP-9, and VEGF levels in culture supernatants of HONE-1 cells, which could be inhibited by the beta-blocker propranolol. Norepi induced the invasiveness of all NPC cell lines in a dose-dependent manner, which was blocked by CMT-3, an MMP inhibitor, and propranolol. Norepi stimulated the release of functional angiogenic VEGF by HONE-1 cells as well. Finally, HONE-1 cells were shown to express beta-adrenergic receptors as did seven of seven NPC biopsies examined. The data suggest that catecholamine hormones produced by the sympathetic-adrenal medullary axis may affect NPC tumor progression, in part, through modulation of key angiogenic cytokines.

Rho Kinase Mediates Cold-Induced Constriction of Cutaneous Arteries Role of α2C-Adrenoceptor Translocation

Cold-induced vasoconstriction in cutaneous blood vessels is mediated in part by increased activity of vascular smooth muscle &agr;2-adrenoceptors (VSM &agr;2-ARs). In mouse cutaneous arteries, &agr;2C-ARs are normally silent at 37°C but mediate cold-induced augmentation of &agr;2-AR responsiveness. In transfected HEK293 cells, this functional rescue is mediated by cold-induced translocation of &agr;2C-ARs from the Golgi to the plasma membrane. Experiments were performed to determine the role of Rho/Rho kinase signaling in this process. Inhibition of Rho kinase (fasudil, Y27632 or H-1152) did not affect constriction of isolated mouse tail arteries to the &agr;2-AR agonist UK 14 304 at 37°C but dramatically reduced the augmented responses to the agonist at 28°C. After Rho kinase inhibition, cooling no longer increased constriction evoked by &agr;2-AR stimulation. Cooling (to 28°C) activated Rho in VSM cells and increased the calcium sensitivity of constriction in &agr; toxin-permeabilized arteries. Stimulation of &agr;2-ARs in VSM cells had no effect on Rho activity or calcium sensitivity at 37°C or 28°C. In HEK293 cells transfected with &agr;2C-ARs, cooling (to 28°C) stimulated the translocation of &agr;2C-ARs to the plasma membrane and this effect was prevented by inhibition of Rho kinase, using fasudil or RNA interference. Consistent with inhibition of the spatial rescue of &agr;2C-ARs, fasudil inhibited &agr;2-AR–mediated mobilization of calcium in tail arteries at 28°C but not 37°C. Therefore, cold-induced activation of Rho/Rho kinase can mediate cold-induced constriction in cutaneous arteries by enabling translocation of &agr;2C-ARs to the plasma membrane and by increasing the calcium sensitivity of the contractile process.

Increased α2-adrenergic constriction of isolated arterioles in diffuse scleroderma

OBJECTIVE Vasospasm and ischemic organ injury are important in the pathogenesis of systemic sclerosis (SSc; scleroderma). The present study was performed to determine whether SSc arterioles have an intrinsic disturbance in vasoconstrictor activity. METHODS Skin biopsy samples were obtained from the upper arm of 11 patients with diffuse SSc (clinically uninvolved skin) and 8 age- and sex-matched control subjects. Dermal arterioles were dissected from the biopsy sample and mounted in a myograph for continuous monitoring of arteriolar diameter. The resting internal diameter of control and SSc arterioles was similar (mean +/- SEM 164+/-15 micro and 166+/-18micro, respectively). RESULTS Dermal arterioles displayed no spontaneous constrictor activity in the absence of stimulation. Vasoconstriction in response to KCI, a receptor-independent activator of smooth muscle, or to phenylephrine, a selective alpha1-adrenergic receptor (alpha1-AR) agonist, was similar in control and SSc arterioles. However, constrictor responses to UK 14,304, a selective alpha2-AR agonist, were increased in SSc compared with control arterioles (maximal constriction responses of 25+/-5% and 67+/-4% [mean +/- SEM] in control and SSc arterioles, respectively; P = 0.000014). Mechanical denudation of the endothelium did not alter reactivity to alpha2-AR activation, indicating that the enhanced constriction in SSc was not mediated by changes in endothelial dilator activity. Indeed, in arterioles constricted with phenylephrine, the endothelial stimuli acetylcholine or bradykinin evoked endothelium-dependent relaxation that was similar in control and SSc arterioles. CONCLUSIONS Vascular smooth muscle in SSc arterioles displayed a selective increase in alpha2-AR reactivity. The endothelial dilator function appeared normal. Altered activity of smooth muscle alpha2-ARs may contribute to the vasospastic activity that is a prominent feature of the SSc disease process.

The impact of cyclooxygenase-2 mediated inflammation on scarless fetal wound healing

Cyclooxygenase-2 (COX-2) and the prostaglandin products generated as a result of COX-2 activity mediate a variety of biological and pathological processes. Scarless healing occurs in fetal skin in the first and second trimesters of development. This scarless healing process is known to proceed without a significant inflammatory response, which appears to be important for the lack of scarring. Because the COX-2 pathway is an integral component of inflammation, we investigated its role in the fetal repair process using a mouse model of scarless fetal wound healing. COX-2 expression in scarless and fibrotic fetal wounds was examined. In addition, the ability of exogenous prostaglandin E(2) to alter scarless fetal healing was evaluated. The results suggest that the COX-2 pathway is involved in scar production in fetal skin and that targeting COX-2 may be useful for limiting scar formation in adult skin.

The vasculopathy of Raynaud's phenomenon and scleroderma.

The scleroderma (SSc) disease process involves dramatic dysfunction in acute and chronic vascular regulatory mechanisms; it presents initially with heightened vasoconstrictor or vasospastic activity and progresses to structural derangement or vasculopathy of the microcirculation. This article discusses the regulatory mechanisms that contribute to this dysfunction and the vascular changes in the context of the other aspects of the SSc disease process in a novel attempt to integrate the individual pathologies of the disease process.

Increased Expression of Cyclooxygenase-2 Mediates Enhanced Contraction to Endothelin ETA Receptor Stimulation in Endothelial Nitric Oxide Synthase Knockout Mice

The aim of this study was to determine whether prolonged loss of NO activity, in endothelial NO synthase knockout (eNOS−/−) mice, influences endothelin (ET) ETA receptor–mediated smooth muscle contraction and, if so, to define the underlying mechanism(s). In isolated endothelium-denuded abdominal aortas, contractions to the selective ETA receptor agonist ET-1(1-31) were significantly increased in aortas from eNOS−/− compared with wild-type (WT) mice. In contrast, contractions to the &agr;1-adrenergic agonist phenylephrine or the thromboxane (TX) A2 analog U-46619 were similar between eNOS−/− and WT mice. Immunofluorescent and Western blot analysis demonstrated that the aortic expression of ETA receptors was decreased in eNOS−/− compared with WT mice. Contractions evoked by ET-1(1-31), but not phenylephrine, were reduced by inhibition of cyclooxygenase-2 (COX-2) (indomethacin or celecoxib) or of TXA2/prostaglandin H2 receptors (SQ-29548). After COX inhibition, contractions to ET-1(1-31) were no longer increased and were actually decreased in eNOS−/− compared with WT aortas. Western blot analysis revealed that endothelium-denuded abdominal aortas express COX-2, but not COX-1, and that expression of COX-2 was significantly increased in eNOS−/− compared with WT mice. Contractions to the COX substrate arachidonic acid were also increased in eNOS−/− aortas. Furthermore, ET-1(1-31) but not phenylephrine stimulated production of the TXA2 metabolite TXB2, which was increased in eNOS−/− compared with WT aortas. Therefore, COX-2 plays a crucial and selective role in ETA-mediated smooth muscle contraction. Furthermore, COX-2 expression is increased in eNOS−/− mice, which overcomes a reduced expression of ETA receptors and enables a selective increase in contraction to ETA receptor stimulation.

The effect of phenotype on mechanical stretch-induced vascular smooth muscle cell apoptosis

The present study evaluated mechanical stretch-induced apoptosis in swine vascular smooth muscle cells (VSMC) of different phenotypes. We demonstrated that differentiated VSMC express a greater level of Bcl-2-associated death factor (BAD) and have a significant cell loss when exposed to mechanical stretch (10% elongation, 1 Hz) for 24 h. We further demonstrated that apoptosis was significantly increased only in differentiated VSMC exposed to mechanical stretch. To test the hypothesis that the intracellular level of BAD in VSMC determines its response to mechanical stretch-induced apoptosis, we examined whether BAD expression was upregulated by mechanical stretch-induced apoptosis and was associated with the increase in the apoptosis level of differentiated VSMC. When exposed to mechanical stretch, the expression of BAD in differentiated VSMC was elevated at 1 h and remained at higher levels during the application of stretch (24 h). In contrast, Bcl-2 expression was suppressed during the application of stretch. Moreover, the proapoptotic function of BAD was inhibited by overexpression of Bcl-2 through transient transfection of VSMC with pCEP4-Bcl-2 or incubation of VSMC with vascular epithelial growth factor. These results suggest that mechanical stretch-induced VSMC apoptosis is phenotype dependent. The higher levels of apoptosis of differentiated VSMC upon mechanical stretch were, at least in part, dependent on their intrinsic level of BAD.

Superoxide anion scavengers restore NO-mediated pulmonary vasodilation after lung transplantation

Left lung autotransplantation (LLA) results in a chronic attenuation in endothelium-dependent, nitric oxide (NO)-mediated pulmonary vasodilation. We tested the hypothesis that this abnormality involves a decrease in the effective concentration of NO due to inactivation by superoxide anion. Size- and position-matched pulmonary arterial rings were isolated from the right (control) and left (LLA) lungs of seven dogs 1-5 mo post-LLA. The rings were suspended for isometric tension recording and contracted with phenylephrine, and cumulative dose-response curves for ACh or calcium ionophore (A-23187) were generated. Endothelium-dependent relaxation to ACh was inhibited post-LLA, with the maximum vasorelaxation response reduced from 88 +/- 5 to 63 +/- 5% (P < 0. 01) post-LLA. In contrast, after pretreatment with the superoxide anion scavengers tiron or superoxide dismutase (SOD), the dose-response relationships for ACh were similar in control and LLA rings. Oxypurinol, which inhibits superoxide anion production by endothelial xanthine oxidase, also restored the vasorelaxation response to ACh in LLA rings. The pulmonary vasorelaxant response to A-23187 was also attenuated (P < 0.01) post-LLA, and this effect was entirely reversed by pretreatment with tiron, SOD, or oxypurinol. These results indicate that the attenuated responses to these pulmonary vasorelaxants post-LLA involve inactivation of NO by superoxide anion generated by endothelial xanthine oxidase.

Fas-mediated apoptosis in accelerated graft arteriosclerosis

Pathological conditions have been recognized where vessel destruction is a prominent feature of the pathogenic process. One such condition consists of the chronic rejection of blood vessels in transplanted solid organs. Accelerated graft arteriosclerosis (AGA) is a multifactorial process characterized by the concentric proliferation of smooth muscle cells (SMCs) within the intima of the vessel wall of transplanted organs. Proliferation of SMCs within the intima corresponds to a response of these cells to injury. In situations like restenosis post-angioplasty, the mechanism of injury: the mechanical disruption of the tunica media, is evident. However, in the case of AGA, the mechanism of injury has remained elusive. In this report, we provide evidence that injury to SMCs in AGA vessels requires an intact Fas pathway. The resulting damage to the tunica media and internal elastic lamina, in turn, might trigger the proliferation of intimal smooth muscle cells that appear to be less sensitive to Fas mediated killing, particularly when supported by a favorable context of inflammatory cytokines and growth factors, as it is the case in AGA. This pathogenic process results in a absolute loss of functional blood vessels that is not being compensated by an efficient angiogenic response.

Endothelial defect mediates attenuated vasorelaxant response to isoproterenol after lung transplantation

We have previously demonstrated that pulmonary vasodilation in response to isoproterenol is attenuated in conscious dogs after left lung autotransplantation (LLA). Our present goal was to identify the cellular mechanism responsible for this dysfunction. Size- and position-matched pulmonary arterial rings were isolated from the right (control) and left (LLA) lungs of 23 dogs 1-14 mo post-LLA. The rings were suspended for isometric tension recording and precontracted, and the vasorelaxant responses to activators of the beta-adrenoreceptor signaling pathway were examined. With the endothelium intact the maximal pulmonary vasorelaxant response to isoproterenol was reduced (P < 0.02) to 57 +/- 9% in LLA rings, compared with 87 +/- 3% in control rings. Responses to the Gs protein activator cholera toxin were also attenuated post-LLA, with the concentration-effect curve shifted to the right (P < 0.01) and no change in the maximal response. In contrast, the vasorelaxant responses to forskolin (adenylyl cyclase activator) or dibutyryl cAMP were similar in endothelium-intact control and LLA rings. In endothelium-denuded rings the maximal vasorelaxant responses to isoproterenol were reduced (P < 0.01) to approximately 25% in both control and LLA rings. In denuded rings cholera toxin, forskolin, and dibutyryl cAMP caused 100% vasorelaxation, and the IC50 values for these agonists were similar in control and LLA rings. Isoproterenol increased (P < 0.05) tissue cAMP to the same extent in control and LLA rings with or without endothelium. In contrast, isoproterenol increased (P < 0.05) tissue cGMP only in endothelium-intact rings, and this effect was reduced (P < 0.05) approximately 50% in LLA rings compared with control. Oxypurinol (endothelial xanthine oxidase inhibitor) restored the pulmonary vasorelaxant response to isoproterenol in endothelium-intact LLA rings. Our results provide the first evidence that activation of the beta-adrenoreceptor signaling pathway in endothelium-intact pulmonary arterial rings results in an increase in cGMP. Moreover, the attenuation in beta-adrenoreceptor-mediated pulmonary vasorelaxation post-LLA is due to inactivation of nitric oxide by endothelium-derived superoxide anion.