Thesis (Ph. D.)--Lehigh University, 2001. / Includes vita. Includes bibliographical references (leaves 127-165).

published_or_final_version / Pharmacology / Master / Master of Medical Sciences

published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Endothelial senescence represents one of the major characteristics of vascular aging contributing to the development of cardiovascular diseases. SIRT1 is a NAD+-dependent enzyme catalyzing the deacetylation reaction of various signaling molecules and exerts beneficial effects against aging-associated pathologies. SIRT1 is a potent regulator antagonizing endothelial senescence. Both expression and activity of SIRT1 are down-regulated in senescent endothelial cells. However, the molecular mechanisms underlying the loss-of-SIRT1 function during the occurrence of endothelial senescence remain unknown. The present study reveals that phosphorylation at serine 47(S47) contributes to the loss-of-SIRT1 function during endothelial senescence. In both replicative and premature senescent endothelial cells, increased phosphorylation of SIRT1 at S47 was closely associated with the severity of cellular senescence. Replacing serine 47 residue with a phospho-mimicking aspartic acid residue impaired the anti-senescence activity of this protein. In addition, phosphorylation of SIRT1 at serine 47 inhibited its nuclear-cytoplasmic shuttling and protein-protein interactions with LKB1, a senescence-promoting kinase and telomeric repeat-binding factor 2–interacting protein 1, a telomere and inflammation regulator. As a result, the anti-inflammatory function of SIRT1 was also abolished by phosphorylation at serine 47. Cyclin dependent kinase 5 (CDK5) was identified as an upstream kinase responsible for phosphorylation of SIRT1 at serine 47. During the endothelial senescence, the activity of this kinase was up-regulated which was attributed to the augmented P25, a regulatory subunit of CDK5. Inhibition of this kinase by roscovitine, a CDK5 inhibitor, decreased the phosphorylation of SIRT1 at serine 47, reduced cellular senescence, promoted the cytoplasmic translocation of SIRT1 and attenuated the inflammation in endothelial cells triggered by tumor necrosis factor α. Moreover, the kinase activity of CDK5 was significantly elevated in aorta tissues of apolipoprotein E–deficient mice. Chronic administration of roscovitine alleviated endothelial senescence, vascular inflammation and the development of arterial atherosclerosis. These results collectively suggest that CDK 5 is responsible for the phosphorylation of SIRT1 at serine 47, which impairs the anti-senescence activity of enzyme and contributes to loss-of-SIRT1 function during vascular aging. By inhibiting this kinase, SIRT1 function can be improved, in turn preventing the development of endothelial senescence and slowing down the process of vascular aging. In addition to phosphorylation, I have also performed a preliminary study on the ubiquitination of SIRT1. The results demonstrated that SIRT1 ubiquitination was mediated by a Cullin-1-RING E3 ligase complex. Knocking down of cullin-1 enhanced SIRT1 protein expression, promoted proliferation and inhibited senescence in endothelial cells. This discovery may provide novel insights on the anti-vascular aging therapeutic development based on SIRT1 modification. / published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

This thesis investigates the involvement of the non-neuronal cholinergic system in endothelium-dependent relaxations and the impact of hypertension on the function of this system. In Study1 the contribution of nicotinic receptors (nAChRs) to endothelium-dependent relaxations evoked by acetylcholine was examined. Both muscarinic (mAChRs) and nAChR were expressed in the aortic endothelium of spontaneously hypertensive (SHR)and Wistar-Kyoto rats (WKY). However, isometric tension measurements showed that, the muscarinic antagonist atropine abolished the relaxations to acetylcholine in WKY aortae, but only partially inhibited those in SHR aortae. While the nicotinic antagonist mecamylamine inhibited the remaining response in SHR aortae, it did not significantly affect the response solely in either SHR or WKY preparations. Hence, nAChRs mediate endothelium-dependent relaxations to the acetylcholine only in the SHR aorta and only when mAChRs are inhibited. Nicotine, the prototypical nicotinic agonist, also induced endothelium-dependent relaxations in both SHR and WKY aortae which were due to activation of α7-nAChRsbut not by mecamylamine-sensitive α3-nAChR. The acetylcholine-induced, atropine-insensitive relaxations and that to nicotine both involve the PI3K pathway. Thus, activation of nAChRscan contribute to acetylcholine-induced endothelium-dependent relaxations via PI3K signaling pathway in aortae of hypertensive animals. Study 2 examined the involvement of non-neuronal cholinergic system in endothelium-dependent relaxations. Isometric tension measurements showed that mild hypothermia (37℃–31℃) induced endothelium-dependent relaxations, which were reduced by atropine, tubocurarine, acetylcholinesterase (enzyme responsible for acetylcholine degradation), bromoacetylcholine (inhibitor of acetylcholine synthesis), hemicholinium-3 (inhibitor of choline uptake) and vesamicol (inhibitor of acetylcholine release) in SHR but not in WKY aortae, indicating that the non-neuronal cholinergic system is involved in mild hypothermia-induced endothelium-dependent relaxations. Compared with WKY, SHR preparations expressed similar levels of acetylcholinesterase and choline acetyltransferase, but lesser vesicular acetylcholine transporter, located mainly in the endothelium. A choline/acetylcholine assay showed that, mild hypothermia increased the uptake of choline by the endothelium of SHR,but not WKY, aortae from extracellular environment for acetylcholine production. To define possible different mechanisms employed by SHR and WKY endothelial cells, the involvement of transient receptor potential (TRP)channels in mild hypothermia-induced response were examined using selective pharmacological inhibitors of different subtypes of TRP channels, namelyAMTB (TRPM8 antagonist),HC-030031 (TRPA1 antagonist)and HC-067047 (TRPV4 antagonist).The results suggest that both TRPM8 and TRPA1 play a role in the response to mild hypothermia in the WKY aorta; however, in the SHR aortaTRPV4,but not TRPA1, channels are activated by mild hypothermia. Moreover, the observation that the mild hypothermia-induced increases in cyclic guanosine monophosphate (cyclic GMP)and choline uptake were inhibited by HC-030031 in WKY but by HC-067047 in SHR aortae further indicate that in the hypertensive strain compensatory TRPV4 activation can make up for the loss of TRPA1-mediated NO production, and that the endothelial cells of the hypertensive animal utilize TRPV4 channels to activate the production of endogenous acetylcholine in response to mild hypothermia. Taken in conjunction, the results reported in this thesis together suggest that hypertension alters the function of the non-neuronal cholinergic system (e.g. n-AChR sensitivity or acetylcholine production) to modulate endothelium-dependent relaxations. / published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

The primary functions of the cardiovascular system are to transport the required nutrients to sustain the metabolic activity of tissues and to remove the metabolic waste products from tissue. Therefore, a steady supply of oxygen for the cells is crucial. Vascular endothelium synthesizes both relaxing and contracting factors to regulate the local tissue blood flow. Nitric oxide (NO) is one of the major endothelium-derived relaxing factor. It is synthesized by endothelial nitric oxide synthase (eNOS), which interacts with soluble guanylyl cyclase in smooth muscle to produce cyclic guanosine monophosphate (cGMP) resulting in vascular relaxation. Nitroglycerin (GTN) is an organic nitrate used for the management of angina pectoris, hypertension and congestive heart failure, it is converted to NO or closely related moleculesin the vasculature resulting in vasodilatation. It has been demonstrated that chronic administration of GTN leads to nitrate tolerance, which is associated with increased arginase activity and reactive oxidative species (ROS)production. Supplement of L-arginine appears to reduce nitrate tolerance, however, the bioavailability of L-arginine is limited. It has been reported that L-citrulline supplement in human results in elevated plasma level of L-arginine. This study was designed to investigate the effect of long-term GTN treatment on the expressions and activities of arginase I, arginase II and eNOS, and on the production of cGMP inhuman umbilical vein endothelial cells (HUVECs).Moreover, the effect of L-citrulline on nitrate tolerance due to GTN treatment was examined. HUVECs were treated with GTN (10 µM), thrombin, L-arginine, L-citrulline and L-norvaline, alone or in combinations, for 1 hour (short-term treatment) or 24 hours(long-term treatment) followed by 30 minutes stimulation by GTN (100 µM).Western immunoblotting was used to measure the protein expression levelsof eNOS, arginase I and arginase II. Enzyme immunoassay and colorimetric assay were performed to determine the cGMP level and the arginase activities, respectively. Our results suggested that the amount of cGMP release in response to acute (30 minutes) GTN stimulation (100 µM)was reduced in HUVECs that were pre-treated with24 hour GTN(10 µM), and this is associated with an increased arginase activity. L-arginine,L-citrullineor L-norvaline alone was not able to prevent this reduction in cGMP release (nitrate tolerance), although L-arginine and L-norvaline, but not L-citrulline, prevented the increase in arginase activity. The combination of L-arginine or L-citrulline with L-norvaline is effective to protect HUVECs against nitrate tolerance. On the other hand, thrombin (a stimulator of eNOS, 1 U/ml) pre-treatment for 24 hours also reduced cGMP production toacute GTN stimulation. Similar to long-term GTN-induced nitrate tolerance, long-term thrombin-induced nitrate tolerance was not prevented by concomitant presence of L-arginine, L-citrulline or L-norvaline. Long-term thrombinpre-treatment also increased arginase activity and this effect was inhibited by L-arginine, L-citrulline and L-norvaline. The increased arginase activity by long-term GTN or thrombin pre-treatment was not associated with increased arginase protein expression. In conclusion, the present data suggested that prolonged pre-exposure to exogenous GTN or endogenous increase NO by thrombin induced nitrate tolerance in HUVECs, and this is unlikely the consequence of the increased arginase activity. In addition, neither L-arginine nor L-citrulline were effective in protecting HUVECs against nitrate tolerance, and only the combination of L-citrulline and L-norvaline or L-arginine and L-norvaline were effective. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Medical Sciences

Some stem-like cells that have received limited attention but may be of considerable clinical value reside in the transition zone between the corneal endothelium (CE) and trabecular meshwork (TM) at the posterior limbus of the eyes. A growing body of evidence has revealed that these cells may be able to rejuvenate the CE, TM or both. However, these stem-like cells have not been clearly defined and we have thus named them PET cells (Progenitor cells of the Endothelium and Trabeculum). Both the CE and TM cells are unique cell types in the eye that do not normally replace themselves once they are lost in ageing or diseases, such as Fuch’s endothelial dystrophy and primary open angle glaucoma. To date, no therapy exists that targets the rejuvenation of the compromised CE or TM in diseases. Therefore, the potential to repair or replace the diseased CE or TM through a cell repopulation approach is an important area that needs to be explored. The use of PET cells for tissue rejuvenation may represent an attractive therapeutic direction for the regeneration of the CE and/or TM. As a good animal model for PET cells is lacking, we sought to identify whether bovine eyes could serve as a good large tissue model for the studies of PET cells. The comparative anatomy of the human and bovine posterior limbus was studied using light, confocal and scanning electron microscopy. Immunohistochemical studies were performed to localize the stem cell niche for the PET cells. Sphere culture was used to isolate and amplify progenitor cells from the CD and TM respectively. A detailed characterization of the spheres and their progenies was performed with immunocytochemistry, quantitative reverse-transcription polymerase chain reaction and differentiation and functional assays. We showed the presence of stem or progenitor cells in the bovine CE, transition zone and TM in situ. The bovine TM insert region may house a stem cell niche that is comparable to that observed in humans. The progenitor cells isolated from the bovine CE and TM that grew as floating spheres demonstrated similar phenotypes in terms of stem cell marker expression. In addition, both the CE and TM spheres were bipotent, highly proliferative and had limited self-renewal capacity. However, they showed a high propensity to differentiate back into the cell type of their tissue of origin. We speculated that the PET cells become more tissues-specific as they migrate away from their niche towards the CE or TM. This may be a reasonable explanation why the CE and TM spheres respectively adopted their original lineage upon non-directed differentiation. Taken together, our results support the hypotheses that PET cells are present in the posterior limbus of bovine eyes and that bovine eyes may serve as a good large tissue model for the studies of these cells. The PET cells represent an attractive target for developing new treatments to regenerate both the CE and TM, thereby reducing the requirement for donor corneas and invasive treatments in glaucomatous patuents. / published_or_final_version / Ophthalmology / Doctoral / Doctor of Philosophy

In blood vessel of mature animal, endothelial cells remain quiescent for years, before apoptosis and being replaced by newly generated endothelial cells. During aging, this turnover process is accelerated and the fast generated endothelial cells become dysfunctional. Endothelial dysfunction in blood vessel is characterized by the imbalanced production of endothelium-dependent relaxing factors (EDRF) and endothelium-dependent contracting factors (EDCF). The NAD-dependent deacetylase SIRT1 is an anti-aging protein with therapeutic potential for aging related cardiovascular diseases. Endothelium-specific over-expression of human SIRT1 promotes endothelium-dependent vasodilatation and endothelium-selective inhibition of human SIRT1 inhibit it. It is accepted that SIRT1 plays a protective role in endothelium dysfunction. However, the underlying mechanisms remain unclear. In the present study, the endothelial functions of a transgenic mouse model with endothelium-selective over-expression of human SIRT1 (hSIRT1) were evaluated and compared with those of wild type mice. Aging-induced deterioration in endothelium-dependent vasodilatation was observed in wild type but not hSIRT1 mice. Endothelium-specific over-expression of SIRT1 prevented aging-induced reduction of NO bioavailability in aortae, without changing endothelial nitric oxide synthase (eNOS) expression levels. Enhanced phosphorylation of eNOS at serine 1177 was detected in hSIRT1 mice aorta. In the presence of Nω-Nitro-L-arginine methyl ester (L-NAME), the nitric oxide synthase (NOS) inhibitor, EDCF induced contraction to acetylcholine was significantly decreased in carotid arteries of hSIRT1 mice. Cyclooxygenase-2 (COX-2) expression was induced by aging in wild-type mice but not in hSIRT1 mice. Thus, both the augmented NO bioavailability and the reduced production of COX-2-derived EDCF in hSIRT1 mice enhanced their endothelial function. To further explore the eNOS-independent mechanism underlying the vasoprotective role of SIRT1, the eNOS deficient and hSIRT1 endothelium-specific over-expression (eNOS-hSIRT1) mice was generated. Decreased endothelium-dependent contraction to acetylcholine was observed in both the carotid artery and aorta of eNOS-hSIRT1 mice, when compared to the controlled eNOS deficient mice. Besides, ATP induced endothelium-dependent contraction, which was COX-dependent, was also decreased in aortae of eNOS-hSIRT1 mice. Thus, the improved endothelial function induced by the endothelium-selective overexpression of SIRT1 was partly attributed to the reduced COX function, independent of eNOS signaling pathway. In summary, endothelium-selective overexpression of human SIRT1 prevented aging-induced impairment of endothelial function via both eNOS dependent and independent mechanisms. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences