In this paper, we study the industrial production index of Sweden from Jan, 2000 to latest Feb, 2010. We find out there is a structural break at time point Dec, 2007, when the global financial crisis burst out first in U.S then spread to Europe. To model the industrial production index, one of the business cycle indicators which may behave nonlinear feature suggests utilizing a smooth transition autoregressive (STAR) model. Following the procedures given by Teräsvirta (1994), we carry out the linearity test against the STAR model, determine the delay parameter and choose between the LSTAR model and the ESTAR model. The results from the estimated model suggest the STAR model is better performing than the linear autoregressive model.

In this thesis, we will study the smooth linear section of the Grassmannian Gr(2, n). Explicitly, we give a criterion for the rationality of such linear section in terms of its codimension in the Plü ̈cker embedding in projective space. Moreover, to obtain a better understanding of the birational parametrization of these linear sections, we analyze their Hodge structures in the cases of even and odd codimensions. To be more precise, we provide numerous examples which suggest certain patterns of Hodge diamonds corresponding to even and odd cases and derive the proof of general patterns for codimension 3 smooth linear section of Gr(2, n) corresponding to odd and even n. / page 29 is missing from hardcopy

The effect of Ddr1 deletion on the expression of genes involved in atherosclerotic vascular remodeling and on the development of atherosclerotic calcification Pamela J. Ahmad, PhD Institute of Medical Science, 2008 During atherosclerosis, collagen molecules, which are abundant in the healthy vessel, are extensively degraded, re-synthesized or newly synthesized, and remodeled to induce profound changes in VSMCs as they colonize and expand atherosclerotic lesions. The central theme of this thesis was to investigate the effect of genetic deletion of a collagen receptor, DDR1, on VSMC processes during atherosclerosis. In the first study, we demonstrated a role for DDR1 as an important regulator of gene expression in synthetic VSMCs. We have profiled the expression of vascular collagen matrix molecules, MMPs and TIMPs in synthetic VSMCs and we have demonstrated that deletion of Ddr1 is sufficient to accelerate ECM remodeling in synthetic VSMCs, which may influence cell migration during atherosclerosis. Moreover, we have extended our knowledge of DDR1 function in synthetic VSMCs, by demonstrating that DDR1 limits VSMC proliferation in a complex matrix microenvironment representative of the ECM produced in the vessel wall during vascular disease. In the second study, we investigated the role of DDR1 in atherosclerotic calcification, a feature of advanced atherosclerotic disease. Here, we demonstrated that intimal calcification in Ldlr-/- mice fed a high-fat/ high-cholesterol diet may be mediated through the initiation of a chondrogenic transcriptional regulatory program and that deletion of Ddr1 significantly attenuated the frequency and extent of atherosclerotic mineralization in vivo, as well as the ability of vascular smooth muscle cells to calcify in vitro, suggesting an important role for DDR1 in VSMCs as a positive regulator of this pathological process. In our third study, we provided evidence of a biochemical association between MMP-2 and DDR1b in VSMCs, which involves a direct interaction between MMP-2 and the extracellular region of the DDR1 receptor. In addition, we reported an association between endogenous MMP-2 and Stat1 in VSMCs, providing a platform for future research to investigate the functional consequences of these novel interactions.

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.

Methylglyoxal (MG) is a reactive á-oxoaldehyde and a glucose metabolite. Previous studies in our laboratory have shown that MG induces the production of reactive oxygen species (ROS), such as superoxide (O2.-), nitric oxide (NO) and peroxynitrite (ONOO-), in vascular smooth muscle cells (VSMCs, A-10 cells). However, the effect of endogenous MG and mechanisms of MG-induced oxidative stress have not been thoroughly explored. The present study investigated fructose (a precursor of MG)- induced ONOO- formation in A-10 cells and whether this process was mediated via endogenous MG formation; roles of MG in regulating mitochondrial ROS (mtROS) production and mitochondrial functions in A-10 cells; and effect of MG on neutrophils in patients with type 2 diabetes mellitus (T2DM). Fructose induced intracellular production of MG in a concentration- and time- dependent manner. A significant increase in the production of NO, O2.−, and ONOO− was observed in the cells exposed to fructose or MG. Fructose- or MG-induced ONOO− generation was significantly inhibited by MG scavengers and by O2.− or NO inhibitors. The data showed that fructose treatment increased the formation of ONOO− via increased NO and O2.− production in A-10 cells, and this effect was directly mediated by an elevated intracellular concentration of MG. By inhibiting complex III and manganese superoxide dismutase activities, MG induced mitochondrial overproduction of O2.-, and mitochondrial ONOO- further. MG also reduced mitochondrial ATP synthesis, indicating the dysfunction of mitochondria. In addition, MG increased plasma NO levels in patients with T2DM, which reflected the oxidative status in those patients. MG-induced oxidative stress in patients with T2DM significantly enhanced levels of cytokines released from neutrophils. Moreover, the neutrophils from T2DM patients showed a greater proclivity for apoptosis, which was further increased by in vitro MG treatment. Our data demonstrate that MG-induced oxidative damage, particularly ONOO- production, contributes to the pathogenesis of T2DM and its vascular complications.

Increased intracellular calcium (Ca2+) is required for vascular smooth muscle cell (VSMC) proliferation through mechanisms that are not well-known. Preventing calmodulin (CaM)-cyclin E interaction with a synthetic peptide inhibits VSMC proliferation in a cyclin E-dependent manner, without increasing de-differentiation or cell death, or affecting re-endothelialization or collagen deposition. Moreover, in situ Ca2+-sensitive phosphorylation and degradation of the cell cycle inhibitor p27Kip1 (p27) in VSMC is specific to G1 and dependent on camodulin kinase-II (CaMK-II) and the proteasome, but not MEK. Lastly, IQGAP1 binding to CaM increases during G1 with no change in total IQGAP1 expression across the cell cycle. Therefore, we determined the clinical potential of an established mechanism (CaM/cyclin E), the existence of a putative mechanism (CaMK-II/p27), and a target novel mechanism (CaM-IQGAP1). Characterization of calcium-sensitive mechanisms of VSMC cycle control could form the basis for new drug-eluting stent agents that have increased selectivity for rapidly dividing VSMC.

Increased intracellular calcium (Ca2+) is required for vascular smooth muscle cell (VSMC) proliferation through mechanisms that are not well-known. Preventing calmodulin (CaM)-cyclin E interaction with a synthetic peptide inhibits VSMC proliferation in a cyclin E-dependent manner, without increasing de-differentiation or cell death, or affecting re-endothelialization or collagen deposition. Moreover, in situ Ca2+-sensitive phosphorylation and degradation of the cell cycle inhibitor p27Kip1 (p27) in VSMC is specific to G1 and dependent on camodulin kinase-II (CaMK-II) and the proteasome, but not MEK. Lastly, IQGAP1 binding to CaM increases during G1 with no change in total IQGAP1 expression across the cell cycle. Therefore, we determined the clinical potential of an established mechanism (CaM/cyclin E), the existence of a putative mechanism (CaMK-II/p27), and a target novel mechanism (CaM-IQGAP1). Characterization of calcium-sensitive mechanisms of VSMC cycle control could form the basis for new drug-eluting stent agents that have increased selectivity for rapidly dividing VSMC.

ATP-sensitive potassium (K+) channels (KATP) in vascular smooth muscle cells (VSMC) play a major role in the regulation of vascular tone by coupling cell contractility and K+ fluxes to cellular metabolism. They are composed of the regulatory sulphonylurea receptors (SUR) and the pore-forming inwardly rectifying K+ (Kir) channels. SUR subunits interact closely with Kir subunits by conferring their sensitivity to nucleotide or sulphonylurea. However, the modulatory mechanisms of KATP channels in VSMC are largely unknown. In particular, the effects of hydrogen sulfide (H2S) and hydroxylamine (HA) on KATP channels and underlying mechanisms have not been addressed in VSMC of resistance arteries. The combined approaches including molecular biology, biochemical assays, and patch-clamp techniques were applied. The electrophysiological and pharmacological features of native KATP channels in VSMC and cloned KATP channels in HEK-293 cells, and the modulation of KATP channels by H2S and HA in single freshly isolated VSMC from rat mesenteric arteries were characterized. In the present study, only small conductance KATP channels of 13 pS were found in rat mesenteric artery VSMC. The recorded macroscopic and unitary KATP currents were activated by nucleoside diphosphate in the presence of magnesium and K+ channel openers, inhibited by a specific KATP channel blocker glibenclamide, but were insensitive to ATP inhibition. The reversal potential shifted rightward in response to the elevation of extracellular K+ and matched the calculated K+ equilibrium potential, indicating the basal currents in both VSMC and HEK-293 cells are carried by K+ ions. Heterologous expression of Kir6.1 with SUR2B in HEK-293 cells formed functional channels and elicited whole-cell K+ currents, which shared some similar biophysical characteristics of native KATP channels in VSMC. Basal KATP currents and resting membrane potential in VSMC were reduced by glibenclamide, demonstrating that KATP channels contribute to background K+ conductance and in the setting of resting membrane potential in this resistance artery. Exogenous H2S enhanced macroscopic and unitary KATP currents with an EC50 of 116 ± 8.3 µM and hyperpolarized membrane potential. H2S activated KATP channels by increasing the open probability of single channels, but not single channel conductance. The reduced endogenous H2S production by D, L-propargylglycine resulted in the attenuation of KATP currents. H2S-induced activation of KATP channels and resultant hyperpolarization were not mediated by cGMP signaling pathway. HA enhanced reversibly KATP currents in a dose-dependent fashion with an EC50 of 54±3.4 µM and also hyperpolarized the cell membrane. HA-stimulated KATP currents were blocked by free radical scavengers (superoxide dismutase and N-acetyl-L-cysteine), and KATP channels were stimulated by a free radical generating system (hypoxanthine/xanthine oxidase), indicating the involvement of superoxide (O2-) in HA effects. Sodium nitroprusside and 8-Br-cGMP did not affect basal KATP currents and HA-stimulated KATP currents, disproving the involvement of NO-sGC-cGMP-mediated signaling pathway in the HA effects. Therefore, HA-induced KATP channel activation and hyperpolarization are likely due to the generation of O2-. In conclusion, KATP channels in resistance artery VSMC serve as the regulatory targets of H2S and HA. These two endogenous molecules modulate KATP channels via different mechanisms. H2S may directly act on KATP channel proteins while HA oxidized them via the formation of O2-, leading to the activation of KATP channels.

Cells in the body respond to mechanical loads in ways that are crucial to normal and disease physiology. Understanding these processes is difficult due to the complex mechanical environment in vivo. In this research, we have developed several cell-stretching devices capable of subjecting cell cultures to non-uniform stretch distributions in order to investigate pathological responses of vascular smooth muscle cells to physiologic stretches. 10T1/2 cells were cyclically stretched with these devices for 24 hours upon silicone membranes, PDMS tubes, and within 3D PEGDA hydrogels. After stretching, altered cell behaviors were measured, including orientation, proliferation (quantified by BrdU incorporation), and gene expression (quantified by real-time, RT-PCR). Cells demonstrated marked changes in orientation, proliferation, and mRNA expression, which all varied with cellular location in the non-uniform environment. More specifically, increased orientation, increased proliferation, and more dramatically altered mRNA expression were found in regions of high, uniaxial stretch, relative to regions of low, near-equibiaxial stretch. These findings demonstrate the capabilities of graded stretch distributions to produce graded cell responses, indicating potentially localized smooth muscle cell behavior in a diseased artery. The novel devices employed herein will hopefully improve our understanding of these complicated cellular pathways, ultimately allowing for improved treatment or prevention of vascular disease.

The effect of Ddr1 deletion on the expression of genes involved in atherosclerotic vascular remodeling and on the development of atherosclerotic calcification Pamela J. Ahmad, PhD Institute of Medical Science, 2008 During atherosclerosis, collagen molecules, which are abundant in the healthy vessel, are extensively degraded, re-synthesized or newly synthesized, and remodeled to induce profound changes in VSMCs as they colonize and expand atherosclerotic lesions. The central theme of this thesis was to investigate the effect of genetic deletion of a collagen receptor, DDR1, on VSMC processes during atherosclerosis. In the first study, we demonstrated a role for DDR1 as an important regulator of gene expression in synthetic VSMCs. We have profiled the expression of vascular collagen matrix molecules, MMPs and TIMPs in synthetic VSMCs and we have demonstrated that deletion of Ddr1 is sufficient to accelerate ECM remodeling in synthetic VSMCs, which may influence cell migration during atherosclerosis. Moreover, we have extended our knowledge of DDR1 function in synthetic VSMCs, by demonstrating that DDR1 limits VSMC proliferation in a complex matrix microenvironment representative of the ECM produced in the vessel wall during vascular disease. In the second study, we investigated the role of DDR1 in atherosclerotic calcification, a feature of advanced atherosclerotic disease. Here, we demonstrated that intimal calcification in Ldlr-/- mice fed a high-fat/ high-cholesterol diet may be mediated through the initiation of a chondrogenic transcriptional regulatory program and that deletion of Ddr1 significantly attenuated the frequency and extent of atherosclerotic mineralization in vivo, as well as the ability of vascular smooth muscle cells to calcify in vitro, suggesting an important role for DDR1 in VSMCs as a positive regulator of this pathological process. In our third study, we provided evidence of a biochemical association between MMP-2 and DDR1b in VSMCs, which involves a direct interaction between MMP-2 and the extracellular region of the DDR1 receptor. In addition, we reported an association between endogenous MMP-2 and Stat1 in VSMCs, providing a platform for future research to investigate the functional consequences of these novel interactions.