The ability to obtain a potentially unlimited source of endothelial cells from hESCs holds great promise for future regenerative medicine therapies. However, stem cell therapy for cardiovascular disease will ultimately be used in patients with risk factors such as tobacco smoking. We therefore were interested whether nicotine, a key component of tobacco smoke, would have any effect on the survival or incorporation of hESC-ECs into the ischemic heart. Surprisingly, short-term exposure to nicotine at clinically relevant concentrations (eg. in the nanomolar range) enhanced hESC-EC survival in the ischemic heart, and new vessel formation. Following intramyocardial injection of DF+hESC-ECs, we observed a rapid decrease in imaging intensity over seven days in the control group, which indicated significant cell death. However, we were surprised to see significant prolongation of DF+hESC-EC survival in the nicotine-treated group out to 5 weeks.
To understand the mechanism of nicotine-induced hESC-EC survival, we next performed a series of in vitro
experiments using different concentrations of nicotine (10−8
M to 10−2
M). Significant cell death occurred at the higher concentrations (10−4
M), but at lower concentrations (10−8
M) we observed increased proliferation, anti-apoptosis, and angiogenesis. In particular, the 10−8
M concentration had the largest effect on proliferation. Our in vitro
analysis suggests that as a non-selective agonist of nAChRs, nicotine (at the clinically relevant dose of 10−8
M) can improve hESC-EC angiogenesis and prevent apoptosis under hypoxia through MAPK and Akt signaling pathways. Furthermore, nAChR activation led to upregulation of VEGF-A and bFGF gene expression and enhanced hESC-EC survival and neovasculature formation after delivery in ischemic heart tissue. Interestingly, the finding that angiogenesis was upregulated after hESC-EC transplantation followed by low-dose nicotine administration suggests that the activation of nAChRs can improve the paracrine effect of hESC-EC on VEGF secretion, which is another benefit from stem cell therapy
One limitation of this study is that DF+hESC-ECs were used for in vivo studies, whereas non-transduced cells were assessed for cell survival and angiogenic effects. However, we have shown that the DF+hESC-ECs cells exhibit similar proliferation, viability, and phenotypic markers as non-transduced cells (, ). This result is in agreement with our previous reports that show that transduced hESCs maintain the pluripotent stem cell phenotype. Therefore, it is likely that both DF+hESC-ECs and non-transduced hESC-ECs respond to nAChR activation in a similar manner.
Our investigation into nicotine's effects on stem cell survival is only the latest study of this drug after decades of research into its cellular and physiologic effects. In recent years, a growing body of evidence indicates that non-neuronal nAChRs, when activated by nicotine, may play a prominent role in endothelial cell survival, proliferation
, and mobilization
, through their angiogenic
, and anti-apoptotic
properties. Along with acetylcholine, nicotine is a ligand for nAChRs, which are cholinergic ion channels found in plasma membranes of many different cell types, primarily neurons. Evidence suggests that non-neuronal nAChRs are involved in the regulation of vital cell functions, such as mitosis, differentiation, organization of the cytoskeleton, cell-cell contact, locomotion, and migration 
. Furthermore, at clinically relevant concentrations of nicotine (i.e., 1-100 nM range experienced by smokers or individuals treated with nicotine), nicotine has been shown to promote angiogenesis in a number of in vivo
settings, including inflammation, wound healing, ischemia, tumor, and atherosclerosis
However, these unexpected effects of nicotine, along with their implications for cell-based therapies, are often countered by other compounds found in tobacco smoke. Though nicotine is a key addictive component of tobacco, it is notable that tobacco smoke consists of more than 4,000 chemicals
, many of them carcinogenic or otherwise toxic. For example, the liquid vapor portion of the smoke aerosol contains the compounds acrolein
, which are known to be cytotoxic and mutagenic, and may account for some of second-hand smoke's toxicities
. Nicotine is thus not the sole or even most important mediator of tobacco's harmful effects, and studies demonstrating its angiogenic and proliferative properties through nAChRs on endothelial cells have hinted at the intriguing and varied bio-activities of this compound.
In summary, this is the first study to investigate the effect of nAChRs on hESC-EC behavior both in vitro and in vivo, as well as the first study to elucidate the relationship between nAChRs activation and the downstream signaling pathways including MAPK, Akt, and HIF-1α. MAPK cascades are well known multi-functional signaling networks that influence cell growth, differentiation, apoptosis, and cellular responses to stress. HIF-1α is an important transcriptional factor that activates the gene expression of growth factors and promotes the expression of several genes which confer hypoxic tolerance through angiogenesis, erythropoeisis, vasodilation, and altered glucose metabolism. We have demonstrated in vitro that the activation of nAChRs by its ligand—a low dose of nicotine—can trigger anti-apoptotic, angiogenic, and proliferative pathway. Furthermore, systemic in vivo administrations of nicotine protected hESC-ECs from acute cell loss after transplantation to ischemic heart tissue. Taken together, we believe the activation of nAchRs has a potential positive role to play in regenerative medicine, and may become valuable for improving hESC-EC survival and, ultimately, therapeutic efficacy.