Previous studies have shown that L-tryptophan treatment has been known to reduce blood pressure (BP) in hypertensive rats. L-tryptophan is converted to serotonin (5-HT), a potent vasoconstrictor agonist. The direct vascular effects of L-tryptophan, an essential amino acid, and the mechanism that contributes to the fall in BP have not been fully explored. The present study aims to examine the direct vascular responses to both D- and L- tryptophan using perfused mesenteric vascular bed, an ex-vivo preparation that represents the resistance function of circulation. Perfusion was maintained at a constant flow rate (5 mL/min) with Krebs buffer (pH 7.4, 37˚C) after isolation from 12 to 14 week old male Sprague-Dawley rats. The basal perfusion pressure (PP) (mean ± SEM) was 27 ± 3 mmHg. Inclusion of D- and L-isomers in the perfusion medium led to concentration-dependent increase in PP. While the maximal response (Emax) was similar, D-tryptophan (EC50: 0.25 ± 0.12* µmol; Emax: 128 ± 8 mmHg) was more potent (lower EC50 value; *p < 0.01) than L-tryptophan (EC50: 0.79 ± 0.30 µmol; Emax: 141 ± 7 mmHg). Inclusion of increasing concentrations (2, 5 and 10 nM) of the 5-HT2A selective antagonist, ketanserin, led to parallel right-ward shifts in the concentration-response curves to D- and L-tryptophan with restoration of their Emax. In contrast, the α1 selective agonist, methoxamine (30 µM), constricted preparations, both D- (IC50: 0.94 ± 0.30* µmol; Imax: 96 ± 2%) and L-tryptophan (IC50: 2.8 ± 1.0 µmol Imax: 88± 1%) evoked concentration-dependent vasodilatation, an effect that was resistant to blockade by either ketanserin or other 5-HT antagonists. Again, D-tryptophan was more potent than L-tryptophan in the presence of 5-HT antagonist (*p < 0.05). Neither the removal of endothelium nor incubation with selective inhibitors of dilatory mediators released from the endothelium, failed to alter the vasodilator responses to D- and L-tryptophan. In potassium chloride depolarized preparations, L-tryptophan evoked an additive vasoconstrictor response. The vasodilator responses to L-tryptophan persisted in the presence of glibenclamide, a KATP channel inhibitor, or tetraethyl ammonium, a BKCa channel inhibitor, or BaCl2, a Kir channel inhibitor, or ouabain, a Na+-K+-ATPase pump inhibitor. These data confirm that the essential amino acid, L-tryptophan, as well as its D-isomer, evoke a biphasic vasoconstrictor and vasodilator responses in the resistance type mesenteric vascular bed. While the vasoconstrictor responses are mediated by activation of vascular 5-HT receptors, the endothelium-independent vasodilator responses are not linked to activation of vascular 5-HT receptors, vascular potassium channels, Na+-K+-ATPase pump or via inhibition of voltage-operated Ca2+-channels. Plasma concentration of L-tryptophan is about 90 - 120 µM. The endothelium/5-HT independent direct vasodilator responses characterized here for the first time could account for the antihypertensive/ BP lowering effect of L-tryptophan reported earlier by other laboratories.

We hypothesized that the interaction between tumor necrosis factor alpha(TNF)/nuclear factor-kappaB (NFkB) via activation of IKK may amplify one anotherresulting in the evolution of vascular disease and insulin resistance associated withdiabetes. The interaction between TNFa and monocyte chemoattractant protein-1 (MCP-1) may contribute to the evolution of vascular inflammation and endothelial dysfunctionin coronary arterioles in type 2 diabetes. To test this hypothesis, endothelium-dependent(ACh) and –independent (SNP) vasodilation of isolated, pressurized coronary arterioles(40-100 μm) from mLeprdb (heterozygote, normal), Leprdb (homozygote, diabetic) andLeprdb mice null for TNF (dbTNF-/dbTNF-) were examined. Although dilation of vesselsto SNP was not different between Leprdb and mLeprdb mice, dilation to ACh was reducedin Leprdb mice. The NFkB antagonist, MG-132, IKK inhibitor, sodium salicylate(NaSal), or Anti-MCP-1 partially restored endothelium-dependent coronary arteriolardilation in Leprdb mice. Protein expression of IKK and IKK were higher in Leprdb thanin mLeprdb mice. The expression of IKK, but not the expression of IKK was increasedin dbTNF-/dbTNF- mice. Leprdb mice showed increased insulin resistance, but NaSal improved insulin sensitivity. Protein expression of TNFa, NFkB, phosphorylation ofIKK and JNK were greater in Leprdb mice, but NaSal attenuated protein expression ofthem in Leprdb mice. The ratio of phosphorylated IRS-1 at Ser307 (pIRS-1)/IRS-1protein expression was elevated in Leprdb mice; both NaSal and JNK inhibitor SP600125reduced pIRS-1/IRS-1 in Leprdb mice. MG-132 or neutralization of TNF reducedsuperoxide production in Leprdb mice. Anti-MCP-1 attenuated superoxide productionand protein expression of nitrotyrosine (N-Tyr), which is an indicator of peroxynitriteproduction, in isolated coronary arterioles of Leprdb mice. Immunostaining resultsshowed that expression of MCP-1 and vascular cellular adhesion molecule-1 (VCAM) isco-localized with endothelial cells and macrophages. Anti-TNFa or anti-MCP-1markedly reduced macrophage infiltration and the number of MCP-1 positive cells.Neutralization of TNFa or anti-MCP-1 reduced the expression of adhesion molecules. Inconclusion, our results indicate that the interaction between NFkB and TNFa signalinginduces activation of IKKb. In addition, TNFa and TNFa-related signaling, includingthe expression of MCP-1 and adhesion molecules, further exacerbates oxidative stressleading to endothelial dysfunction in type 2 diabetes.

The vascular endothelium continually senses and responds to both biochemical and mechanical stimuli to regulate vascular function in health and disease. The purpose of this dissertation was to understand the molecular mechanisms by which endothelial cells (ECs) respond to sphingosine 1-phosphate (S1P) and fluid wall shear stress (WSS) to initiate angiogenesis. To accomplish this, a novel cell culture system was developed to study the combined effects of S1P and WSS on inducing EC invasion into three-dimensional (3-D) collagen matrices. EC invasion required the presence of S1P, with the effects of S1P being enhanced by WSS to an extent comparable with S1P combined with pro-angiogenic growth factor stimulation. The extent of EC invasion depended on the magnitude of WSS in a biphasic manner, with the greatest induction occurring at 5.3 dyn/cm2 WSS. Several proteins have been implicated in EC invasion, including calpain, Akt, vimentin, p21-activated kinase (PAK), and membrane type 1-matrix metalloproteinase (MT1-MMP). Interestingly, activations of calpain and MT1-MMP and phosphorylations of Akt, PAK, and vimentin coincided with, and were required for, S1P- and WSS- induced EC invasion. Further, inhibitors of calpain, MT1-MMP, Akt and PAK all attenuated invasion induced by WSS and S1P. Calpain inhibition reduced Akt phosphorylation, vimentin cleavage, and MT1-MMP membrane translocation, suggesting that calpain regulates MT1-MMP via Akt phosphorylation and vimentin remodeling. Akt inhibition also completely blocked MT1-MMP membrane translocation and decreased phosphorylation of PAK and vimentin. In summary, these results suggest a new molecular pathway by which the combination of S1P and WSS stimulates EC invasion through calpain, Akt, PAK and vimentin to regulate activation and membrane translocation of MT1-MMP in 3-D collagen matrices.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 209-255).

The endothelium is important for the regulation of vascular tone. In diseases like hypertension, the endothelial cells become dysfunctional. This dysfunction is characterized by nitric oxide (NO) deficiency, impairment of endothelium-dependent hyperpolarization (EDH) and the overwhelming production of endothelium-derived contracting factor (EDCF). Heme oxygenase (HO) is the rate-limiting enzyme in the catabolism of heme, producing carbon monoxide(CO), bilirubin and free iron. Up-regulation of the inducible isoform (HO-1) of the enzyme lowers blood pressure in animals. The purpose of the present study was to investigate whether or not up-regulation of HO-1by the pharmacological agent hemin improves endothelial function in arteries of spontaneously hypertensive rats(SHR). Twenty four hours after intraperitoneal injection of hemin (50mg/kg) in 36 weeks old SHR, the expression and activity of HO-1 were augmented, in both the endothelium and vascular smooth muscle. Hemin-treatment potentiated endothelium-dependent relaxations to the muscarinic agonist acetylcholine in both the aorta and the mesenteric artery, whereas the HO inhibitor protoporphyrin IX zinc (II) (ZnPP; 30 mg/kg) prevented the beneficial effect of hemin, suggesting that HO-1 induction improves endothelial function. Hemin-treatment did not augment acetylcholine-induced NO-mediated relaxations, and did not alter the expression level of either phosphorylated eNOS (Ser1177) or total eNOS, suggesting that the improvement of endothelial function by HO-1 induction cannot be attributed to an increased bioavailability of NO. In the mesenteric arteries, hemin treatment potentiated acetylcholine-evoked EDH-mediated relaxations in the presence of L-NAME and indomethacin. The IKCa channel blocker TRAM-34andthe Na+-K+-ATPase blocker ouabain significantly impaired these hemin-potentiated relaxations. NS309-induced TRAM-34-and ouabain-sensitive relaxations were enhanced by hemin-treatment. K+-induced ouabain-sensitive relaxations and the expression of Na+-K+-ATPase were increased by hemin-treatment. Taken in conjunction, these observations imply that the improved EDH-mediated relaxations by HO-1 induction is due to an improvement of IKCa-Na+-K+-ATPase pathway. Treatment with an antioxidant apocynin (50mg/kg) showed a similar effect as hemin, and the combined treatment with hemin and apocynin did not cause a greater improvement. In vitro treatment with bilirubin, enhanced EDH responses and K+-induced ouabain-sensitive relaxations. These observations suggest that the effect of HO-1 induction on EDH-mediated relaxations is possibly due to its antioxidant properties and the production of bilirubin. In the aortae, hemin-treatment reduced endothelium-dependent contractions in response to acetylcholineor to a calcium ionophoreA23187. Production of reactive oxygen species (ROS) was suppressed by hemin-treatment, judging from the results of 2’,7’-dichlorodihydrofluoresein diacetate staining, dihydroethidium staining and lucigenin chemiluminescence, which was attributed to the decreased expressions of NADPH oxidase-2 (Nox2) and cyclooxygenase-1(COX-1). The production of prostacyclin was decreased, which was explained by a lower expression of COX-1. Contractions to vasoconstrictor concentrations of prostacyclin and its mimetic iloprost were attenuated, suggesting that the responsiveness of thromboxane-prostanoid receptors (TP receptors) to prostacyclin was decreased by hemin-treatment. The effects of HO-1 on the suppressed production of ROS and prostacyclin, and the decreased responsiveness of TP receptors, contribute to its inhibitory role on EDCF-mediated response. Thus, up-regulation of HO-1 improves endothelial function in the SHR by potentiating EDH response and impairing EDCF. / published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

The vascular endothelium is a dynamic cellular interface that displays a unique phenotypic plasticity. This plasticity is critical for vascular function and when dysregulated is pathogenic in several diseases. The development of new human endothelial genotype-phenotype studies, personalized vascular medicine efforts and cell based regenerative therapies are limited by the unavailability of patient-specific endothelial cells. Induced pluripotent stem cells (iPSC) offer great promise as a new personalized source of endothelium; however, the reproducibility, fidelity and functionality of iPSC-derived endothelial cells remains poorly understood. / Engineering and Applied Sciences

Background: Currently, the optimal long-term therapy for end stage heart failure is heart transplantation. Cardiac allograft vasculopathy contributes to a significant number of deaths following transplantation. This vasculopathy is related to early endothelial injury sustained at the time of organ transplantation and to persistent endothelial injury as a result of cytotoxic immunosuppression, as well as chronic rejection. Epidermal growth factor-like domain 7 (Egfl7), is expressed in endothelial cells upon arterial injury and may have a role in maintaining vascular endothelial integrity and regeneration following injury. Similarly, novel pharmacologic agents such as Bosentan, an endothelin-1 antagonist, and Cilostazol, a phosphodiesterase 3 inhibitor, have been demonstrated to attenuate calcineurin inhibition induced endothelial dysfunction and neointimal hyperplasia, respectively. We hypothesized that, 1) Egfl7 will attenuate endothelial activation, cell adhesion molecule expression and neutrophil adhesion following simulated ischemia-reperfusion injury or exposure to calcineurin inhibition and that, 2) Bosentan and Cilostazol will inhibit neointimal hyperplasia following endothelial injury in a mouse model of vascular injury. Methods: Human coronary artery endothelial cells were subjected to hypoxia-reoxygenation injury or the calcineurin inhibitors Cyclosporine A and Tacrolimus to examine the effects of Egfl7 on these injury mechanisms. Cell adhesion molecule expression, neutrophil adhesion to endothelial cells, and NF-&kappa;B activation were measured. Cell adhesion molecule and Egfl7 expression were also examined in a mouse model of neointimal. This model was used to examine the effects of Bosentan and Cilostazol on neointimal hyperplasia. Results: Egfl7 had potent anti-inflammatory properties including inhibition of NF-&kappa;B pathway activation, ICAM-1 expression and neutrophil adhesion to injured endothelium. Within vessels exhibiting neointimal hyperplasia, Egfl7 was expressed in regions lacking ICAM-1 expression. Both cilostazol and bosentan attenuated neointimal hyperplasia in isolation as well as during co-treatment with CNI therapies. Conclusions: Egfl7 is an endothelial protective signaling protein with anti-inflammatory properties effective against simulated ischemia-reperfusion injury and calcineurin inhibition mediated injury. Cilostazol and Bosentan are pharmacologic strategies demonstrating efficacy against the development of neointimal hyperplasia. These observations provide a novel therapeutic target and strategies that may be relevant to endothelial protection and prevention of cardiac allograft vasculopathy following heart transplantation.

Background: Currently, the optimal long-term therapy for end stage heart failure is heart transplantation. Cardiac allograft vasculopathy contributes to a significant number of deaths following transplantation. This vasculopathy is related to early endothelial injury sustained at the time of organ transplantation and to persistent endothelial injury as a result of cytotoxic immunosuppression, as well as chronic rejection. Epidermal growth factor-like domain 7 (Egfl7), is expressed in endothelial cells upon arterial injury and may have a role in maintaining vascular endothelial integrity and regeneration following injury. Similarly, novel pharmacologic agents such as Bosentan, an endothelin-1 antagonist, and Cilostazol, a phosphodiesterase 3 inhibitor, have been demonstrated to attenuate calcineurin inhibition induced endothelial dysfunction and neointimal hyperplasia, respectively. We hypothesized that, 1) Egfl7 will attenuate endothelial activation, cell adhesion molecule expression and neutrophil adhesion following simulated ischemia-reperfusion injury or exposure to calcineurin inhibition and that, 2) Bosentan and Cilostazol will inhibit neointimal hyperplasia following endothelial injury in a mouse model of vascular injury. Methods: Human coronary artery endothelial cells were subjected to hypoxia-reoxygenation injury or the calcineurin inhibitors Cyclosporine A and Tacrolimus to examine the effects of Egfl7 on these injury mechanisms. Cell adhesion molecule expression, neutrophil adhesion to endothelial cells, and NF-&kappa;B activation were measured. Cell adhesion molecule and Egfl7 expression were also examined in a mouse model of neointimal. This model was used to examine the effects of Bosentan and Cilostazol on neointimal hyperplasia. Results: Egfl7 had potent anti-inflammatory properties including inhibition of NF-&kappa;B pathway activation, ICAM-1 expression and neutrophil adhesion to injured endothelium. Within vessels exhibiting neointimal hyperplasia, Egfl7 was expressed in regions lacking ICAM-1 expression. Both cilostazol and bosentan attenuated neointimal hyperplasia in isolation as well as during co-treatment with CNI therapies. Conclusions: Egfl7 is an endothelial protective signaling protein with anti-inflammatory properties effective against simulated ischemia-reperfusion injury and calcineurin inhibition mediated injury. Cilostazol and Bosentan are pharmacologic strategies demonstrating efficacy against the development of neointimal hyperplasia. These observations provide a novel therapeutic target and strategies that may be relevant to endothelial protection and prevention of cardiac allograft vasculopathy following heart transplantation.

Pulmonary arterial hypertension (PAH) results from endothelial cell (EC) damage leading to pulmonary vasoconstriction and arteriolar remodeling. Patients with PAH exhibit high pulmonary arterial pressures due to increased pulmonary vascular resistance and die of progressive right-sided heart failure. The pathogenesis of PAH is not completely understood, but involves processes which reflect abnormalities in EC function: an imbalance of vasodilators and constrictors, thrombosis, vascular smooth muscle cell (SMC) hypertrophy and proliferation, and susceptibility to EC apoptosis. Therefore, it is important to investigate possible alterations in the underlying mechanisms that regulate EC structure and function. Krüppel-like factor 2 (KLF2) is a shear-responsive transcription factor, highly expressed in the pulmonary ECs under physiological conditions, and known to maintain EC homeostasis by acting as a master switch for a quiescent profile of EC gene transcription. We hypothesized that Klf2 expression is reduced in models of pulmonary hypertension (PH) and its down-regulation contributes to PH development; conversely, Klf2 overexpression is beneficial, and may represent a novel therapeutic target. The role of KLF2 in PH was characterized in two experimental rat models: the monocrotaline model of severe and lethal PAH, and the chronic hypoxia model of reversible hypoxic PH. In vivo Klf2 expression was manipulated using jetPEI® to enhance or reduce the activity of the KLF2 pathway. Plasmids containing short hairpin Klf2 (shKLF2) or Klf2, or empty plasmids were selectively delivered to the pulmonary microvasculature, and the effect on pulmonary hemodynamics, microvascular structure and function, along with various in vitro functional and molecular assays of EC activity, were assessed. Results suggest that reduced Klf2 expression may be a critical early event in EC activation and initiation of PAH; and, its persistent downregulation may play a role in the transition to a progressive and irreversible process. Data also suggests that an early therapeutic intervention to overexpress Klf2, can prevent the development of PH in both models tested when applied before the “irreversible” microvascular remodeling is present. However, once the full PAH phenotype is established, in particular in the presence advanced arteriolar remodeling, Klf2 gene transfer was unsuccessful in reversing the disease in the MCT model.

Pulmonary arterial hypertension (PAH) results from endothelial cell (EC) damage leading to pulmonary vasoconstriction and arteriolar remodeling. Patients with PAH exhibit high pulmonary arterial pressures due to increased pulmonary vascular resistance and die of progressive right-sided heart failure. The pathogenesis of PAH is not completely understood, but involves processes which reflect abnormalities in EC function: an imbalance of vasodilators and constrictors, thrombosis, vascular smooth muscle cell (SMC) hypertrophy and proliferation, and susceptibility to EC apoptosis. Therefore, it is important to investigate possible alterations in the underlying mechanisms that regulate EC structure and function. Krüppel-like factor 2 (KLF2) is a shear-responsive transcription factor, highly expressed in the pulmonary ECs under physiological conditions, and known to maintain EC homeostasis by acting as a master switch for a quiescent profile of EC gene transcription. We hypothesized that Klf2 expression is reduced in models of pulmonary hypertension (PH) and its down-regulation contributes to PH development; conversely, Klf2 overexpression is beneficial, and may represent a novel therapeutic target. The role of KLF2 in PH was characterized in two experimental rat models: the monocrotaline model of severe and lethal PAH, and the chronic hypoxia model of reversible hypoxic PH. In vivo Klf2 expression was manipulated using jetPEI® to enhance or reduce the activity of the KLF2 pathway. Plasmids containing short hairpin Klf2 (shKLF2) or Klf2, or empty plasmids were selectively delivered to the pulmonary microvasculature, and the effect on pulmonary hemodynamics, microvascular structure and function, along with various in vitro functional and molecular assays of EC activity, were assessed. Results suggest that reduced Klf2 expression may be a critical early event in EC activation and initiation of PAH; and, its persistent downregulation may play a role in the transition to a progressive and irreversible process. Data also suggests that an early therapeutic intervention to overexpress Klf2, can prevent the development of PH in both models tested when applied before the “irreversible” microvascular remodeling is present. However, once the full PAH phenotype is established, in particular in the presence advanced arteriolar remodeling, Klf2 gene transfer was unsuccessful in reversing the disease in the MCT model.