Background-Embryonic

Mutations in bone morphogenetic protein (BMP) receptor II (BMPRII) are associated with pulmonary artery endothelial cell (PAEC) apoptosis and the loss of small vessels seen in idiopathic pulmonary arterial hypertension. Given the low penetrance of BMPRII mutations, abnormalities in other converging signaling pathways may be necessary for disease development. We hypothesized that BMPRII supports normal PAEC function by recruiting Wingless (Wnt) signaling pathways to promote proliferation, survival, and motility. In this study, we report that BMP-2, via BMPRII-mediated inhibition of GSK3-β, induces β-catenin (β-C) accumulation and transcriptional activity necessary for PAEC survival and proliferation. At the same time, BMP-2 mediates phosphorylated Smad1 (pSmad1) or, with loss of BMPRII, pSmad3-dependent recruitment of Disheveled (Dvl) to promote RhoA–Rac1 signaling necessary for motility. Finally, using an angiogenesis assay in severe combined immunodeficient mice, we demonstrate that both β-C– and Dvl-mediated RhoA–Rac1 activation are necessary for vascular growth in vivo. These findings suggest that the recruitment of both canonical and noncanonical Wnt pathways is required in BMP-2–mediated angiogenesis.

Objective— An endothelial nicotinic acetylcholine receptor (nAChR) participates in atherogenesis and tumorigenesis by promoting neovascularization. To date, the mechanisms of nAChR-mediated angiogenesis and their relationship to angiogenic factors, eg, VEGF and bFGF, are unknown. Methods and Results— Nicotine induced dose-dependent human microvascular endothelial cell (HMVEC) migration, a key angiogenesis event, to an extent which was equivalent in magnitude to bFGF (10 ng/mL) but less than for VEGF (10 ng/mL). Unexpectedly, nAChR antagonism not only abolished nicotine-induced HMVEC migration but also abolished migration induced by bFGF and attenuated migration induced by VEGF. Transcriptional profiling identified gene expression programs which were concordantly regulated by all 3 angiogens (nicotine, VEGF, and bFGF), a notable feature of which includes corepression of thioredoxin-interacting protein (TXNIP), endogenous inhibitor of the redox regulator thioredoxin. Furthermore, TXNIP repression by all 3 angiogens induced thioredoxin activity. Silencing thioredoxin by small interference RNA abrogated all angiogen-induced migration while silencing TXNIP strongly induced HMVEC migration. Interestingly, nAChR antagonism abrogates growth factor (VEGF and bFGF)–mediated induction of thioredoxin activity. Conclusions— Nicotine promotes angiogenesis via stimulation of nAChR-dependent endothelial cell migration. Furthermore, growth factor–induced HMVEC migration, a key angiogenesis event, requires nAChR activation—an effect mediated in part by nAChR-dependent regulation of thioredoxin activity.

Pathological angiogenesis contributes to tobacco-related diseases such as malignancy, atherosclerosis and age-related macular degeneration. Nicotine acts on endothelial nicotinic acetylcholine receptors (nAChRs) to activate endothelial cells and to augment pathological angiogenesis. In the current study, we studied nAChR subunits involved in these actions. We detected mRNA for all mammalian nAChR subunits except α2, α4, γ and δ in four different types of ECs. Using siRNA methodology, we found that the α7 nAChR plays a dominant role in nicotine-induced cell signaling (assessed by intracellular calcium and NO imaging, and studies of protein expression and phosphorylation), as well as nicotine-activated EC functions (proliferation, survival, migration and tube formation). The α9 and α7 nAChRs have opposing effects on nicotine-induced cell proliferation and survival. Our studies reveal a critical role for the α7 nAChR in mediating the effects of nicotine on the endothelium. Other subunits play a modulatory role. These findings may have therapeutic implications for diseases characterized by pathological angiogenesis.

These data suggest that endogenous activation of nAChR promotes CNV and that activation of nAChR by nicotine may contribute to the increased incidence of CNV seen in smokers with age-related macular degeneration (AMD). Topically administered mecamylamine could provide an appealing new treatment approach for CNV.

Objectives To determine if chronic nicotine exposure blunts angiogenesis. Background Cholinergic angiogenesis is mediated by an endothelial nicotinic acetylcholine receptor (EC nAChR). Short-term administration of nicotine stimulates angiogenesis via EC nAChRs. The long-term effects of nicotine upon cholinergic angiogenesis are unknown. Methods We exposed C57/Bl6 male mice (n=7 in each group) to nicotine (200μg/ml drinking water) or vehicle for 8 or 16 weeks. Subsequently, hindlimb ischemia was induced by ligation of the left femoral artery. After surgery, animals in the vehicle-treated group were re-randomized to vehicle (Vehicle group) or nicotine for 2 weeks (Acute exposure group); whereas animals that had been previously treated (for 8 or 16 weeks with nicotine) continued to receive nicotine (8WK or 16WK groups). After two weeks, animals were sacrificed for immunohistochemical, gene expression, and angiogenesis studies. Results Capillary density of the ischemic hindlimb was increased by nicotine in naïve animals (Vehicle vs Acute exposure: 2.40±0.09 vs 2.82±0.10 capillaries/myocyte, p<0.05). However, prior exposure to nicotine for 16 weeks (16 WK) abolished the effects of nicotine to increase capillary density in the ischemic hindlimb (Acute vs 16 WK : 2.82±0.10 vs 2.47±0.03 capillaries/myocyte; p<0.05). The impairment of cholinergic angiogenesis was associated with a reduction in nAChR expression and plasma VEGF levels. Chronic exposure to nicotine impaired capillary sprouting of aortic segments ex vivo (vehicle vs 16WK :0.303±0.029 vs 0.204±0.017mm2, p<0.05 n=3) Conclusion The current study shows for the first time that chronic exposure to nicotine impairs cholinergic angiogenesis, an effect mediated by downregulation of the vascular nAChR, and attenuation of nicotine-induced VEGF release. These studies may explain the impairment in angiogenic processes observed in long-term smokers.

Rationale Tobacco use is associated with an increase in white blood cell count (WBC). This association has been attributed to bronchopulmonary inflammation and/or infection. It is not known if nicotine itself may play a role. Objectives We determined if nicotine itself could effect WBC count, and determined if this was due to a direct effect on hematopoietic stem cells (HSC). Methods and Measurements C57Bl6J mice received nicotine orally, and measurements of WBC count; bone marrow and spleen cellularity; and HSC count were made. To determine the functionality of HSCs, irradiated animals received bone marrow transplants from vehicle or nicotine treated mice. Main Results Nicotine increased leukocytes in the peripheral blood, bone marrow and spleen. Peripheral red cell and platelet count were unaffected. Nicotine increased the frequency of HSC in the bone marrow. Isolated long-term HSCs from nicotine-treated mice transplanted into irradiated mice regenerated all hematopoeitic cell lineages, demonstrating functional competence of those HSCs. HSC expressed nicotinic acetylcholine receptors (nAChRs), as documented by FITC-conjugated alpha-bungarotoxin binding. Nicotine increased soluble Kit ligand, consistent with stem cell activation. Conclusions The data suggest a new mechanism for the increased WBC associated with tobacco use. The effect of nicotine to activate hematopoiesis may contribute to tobacco-related diseases.

This study was conducted to determine whether cultured human coronary artery and aorta vascular smooth muscle (VSM) cells express the nuclear transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma); whether the thiazolidinedione troglitazone, a ligand for PPARgamma, would inhibit c-fos expression by these cells; and whether troglitazone would inhibit proliferation and migration induced in these cells by mitogenic growth factors. Using immunoblotting and reverse-transcriptase polymerase chain reaction (RT-PCR) techniques, we show that both human aorta and coronary artery VSM cell lines expressed PPARgamma protein and mRNA for both PPARgamma isoforms, PPARgamma1 and PPARgamma2. Immunocytochemical staining localized the PPARgamma protein primarily within the nucleus. Troglitazone inhibited basic fibroblast growth factor and platelet-derived growth factor-BB induced DNA synthesis in a dose-dependent manner and downregulated the growth-factor-induced expression of c-fos. Troglitazone also inhibited the migration of coronary artery VSM cells along a platelet-derived growth factor-BB concentration gradient. These findings demonstrate for the first time the expression and nuclear localization of PPARgamma in human coronary artery and aorta VSM cells. The data also suggest that the downregulation of c-fos expression, growth-factor-induced proliferation, and migration by VSM may, in part, be mediated by activation of the PPARgamma receptor.