The syndrome known as Gastric Dilation Air Sacculitis (GDAS) has recently been described by Lumsden et al. (2002) for Chinook salmon (Oncorhynchus tshawytscha, Walbaum), in seawater (SW) culture in New Zealand. The syndrome is characterised by distended abdomens, gastric dilation and air sacculitis, increased feed conversion ratios (FCR) and mortality. Consequently, financial returns on affected stocks are greatly reduced. A study into the epidemiology and physiology of the syndrome was initiated, working with the major aquaculture company, The New Zealand King Salmon Company (NZKS). The study revealed causative factors of GDAS. GDAS was experimentally induced only in saltwater by feeding a commercially manufactured low-cohesion pelleted diet. Control groups were fed a different diet with high physical cohesion. Low-cohesion pellets have previously been associated with a high incidence of GDAS in commercial sea cages. These data implicated osmoregulatory stress and physical properties of the feed in GDAS development. In addition, gastrointestinal (GI) physiology in GDAS -affected and -control fish was characterised. The process of GDAS development in O. tshawytscha is characterised by a loss of smooth muscle tone of the stomach as it distends. Laplace's law (P= 2T/r, where P is the distending pressure, T is the tension in the wall and r is the radius of the cylinder) predicts that unless muscle mass increases, the ability of the stomach wall to contract will be lost and consequently a loss of GI motor function will result. Therefore, GI circular smooth muscle integrity in terms of (1) stimulated and maximal contractility, (2) osmoregulatory ability of the intestine and the (3) control of the GI system was studied in pathologically affected (+ve) and unaffected (-ve) smolt. Affected fish showed changes in GI circular smooth muscle function and osmoregulatory dysfunction. Feeding different diets induced distinct gastric evacuation patterns. The intestinal brake hypothesis is presented and argued to be the probable mechanism for GDAS development. GDAS (+ve) serum showed the presence of factors capable of contracting gut smooth muscle. In addition, potential humoral mediators of the intestinal brake in fish were investigated.

Intimal hyperplasia (IH) is characteristic of a cell population increase within the innermost layer of the arterial wall. It is hypothesized that extracellular matrix vascular remodeling secondary vascular injury is dependent upon the Th17 subset of the CD4+ lymphocytes. Male C57BL/6J and FVB/NJ murine strains underwent complete left common carotid artery ligation for periods of 14 and 28 days. A therapeutic simvastatin model was carried out in the FVB/NJ strain and involved a daily subcutaneous injection regimen of 40 mg/kg/mouse beginning 72 hours prior to and daily following a 14 day carotid ligation period. Histological and RT-PCR analysis was carried out with harvested carotid artery samples. The FVB/NJ 14 day and 28 day histological stains of the left common carotid artery following ligation injury developed evident structured and disassembled intimal hyperplasia, respectively. A gene array demonstrated dramatic expression of immune and cytokine transcription markers particularly in the FVB/NJ strain at both ligation time points. IL-17 and IL-6 transcriptional gene expression was upregulated greater than 20-fold in the FVB/NJ 28 day injury model. IL-17 transcription was significantly expressed by a change of 50.06 ± 0.19 (p = 0.004) in this strain at 28 days versus the control. Lastly, the simvastatin treatment model was found to exacerbate the immune response to ligation injury. These results revealed that the immune system elicits a role in the vascular remodeling that potentiates intimal hyperplasia.

Collagens are abundant components of the extracellular matrix in the atherosclerotic plaque. In addition to contributing to lesion volume and mechanical stability, collagens can influence the behavior of macrophages and smooth muscle cells (SMCs) and have profound effects on both inflammation and fibrosis during lesion development. The aim of this thesis was to define a functional role for the discoidin domain receptor 1 (DDR1), a collagen receptor tyrosine kinase, in murine models of atherogenesis. In our first study, using Ddr1+/+;Ldlr-/- and Ddr1-/-;Ldlr-/- mice fed a high fat diet, we identified DDR1 as a novel positive regulator of atherogenesis. Targeted deletion of DDR1 attenuated atherosclerotic plaque development by limiting inflammation and accelerating matrix accumulation and resulted in the formation of macrophage poor, matrix rich lesions. In the second study, we used bone marrow transplantation to generate chimeric mice with a deficiency of DDR1 in bone marrow derived cells and reveal a central role for macrophage DDR1 in atherogenesis. Deficiency of DDR1 in bone marrow derived cells reduced lesion size by limiting macrophage accumulation in the developing plaque. Moreover using BrdU pulse labeling, we demonstrated reduced monocyte recruitment into the early fatty streak lesions of Ddr1-/-;Ldlr-/- mice. In our third study, we again utilized bone marrow transplantation to generate mice with deficiency of DDR1 in the host derived tissues such as the vessel wall and uncovered a distinct role for DDR1 expressed on resident vessel wall smooth muscle cells in the regulation of matrix accumulation and fibrous cap formation during atherogenesis. Deficiency of DDR1 in vessel wall cells resulted in robust accumulation of collagen and elastin and resulted in the formation of larger atherosclerotic plaques, with thick fibrous caps. Taken together, these studies support a critical role for DDR1 in the development of the atherosclerotic plaque. We demonstrate that DDR1 exerts distinct and opposing effects on lesion size by regulating both monocyte recruitment and matrix accumulation. These studies underscore the importance of collagen signaling during atherogenesis, and identify DDR1 as a key transducer; providing signals that regulate both inflammation and fibrosis during atherogenesis.

Passive detrusor smooth muscle exhibits both viscoelastic softening and strain softening. Strain softening is a loss of stiffness following a stretch to a longer length and is reversible upon muscle activation. Because of this behavior, steady state passive force in detrusor is not constant for a given muscle length and can be adjusted by an intracellular mechanism. Thus, passive detrusor exhibits adjustable passive stiffness. Existing three-component mechanical models for muscle, the Kelvin and Voigt, are insufficient to display this characteristic. The goal of this thesis is to develop a new biomechanical model for passive force in detrusor by adding additional elements to the Kelvin or Voigt models. Eight mechanical characteristics of detrusor are identified from the literature and with three new experiments, and a novel adjustable passive stiffness model for smooth muscle is proposed. Simulations are performed to demonstrate that the model qualitatively exhibits each of the eight tissue characteristics.

Discoveries made with KCl-induced contractions have elucidated the more complex reactions involved in GPCRs signaling; once the mechanisms of smooth muscle Ca2+ sensitization and desensitization are fully understood, then the development of advanced treatments for vascular disorders such as hypertension, cerebral and coronary vasospasm, and vascular hyporeactivity following hemorrhagic shock may be possible. Studies have shown that KCl-induced contractions induce Ca2+-sensitization. Therefore, we tested the hypothesis that KCl induced Ca2+-sensitization is due to ROK activation by the increase in [Ca2+]i. To test this hypothesis, rabbit femoral arteries were permeabilized with 20µg/ml α-toxin and 1% Triton X-100 and subjected to different calcium concentrations in the presence or absence of various ROK inhibitors. For a comparison we also used various PKC and MLCK inhibitors and repeated these experiments in intact tissues. We found that either [Ca2+]i alone does not directly activate ROK or the permeabilization technique itself disrupts the normal ROK signaling system. Secondary findings revealed that α-toxin activates PKC pathways; in both chemically permeabilized preparations proteases also appear to be activated and MLCK is the primary kinase responsible for contraction.

Hydrogen sulfide (H2S) is receiving increasing interest, as much as nitric oxide (NO) and carbon monoxide have received previously, to understand its physiological functions as it meets all the criteria to define as a third gasotransmitter. Endogenous synthesis from L-cysteine via cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) and the function of H2S as an inhibitor of smooth muscle contraction in gastrointestinal tract are known. However, the loci of generation and action of H2S, and the mechanism of inhibition of contraction are unknown. Hence, my aims in the present study are to: i) identify the expression of enzymes in smooth muscle, ii) determine the effects of endogenously released and exogenously applied H2S on smooth muscle function; and iii) identify the targets and mechanism involved in mediating the effects of H2S using isolated smooth muscle cells from rabbit colon. I have identified the expression of CSE, but not CBS, in smooth muscle and demonstrated that L-cysteine (an activator of CSE) and NaHS (H2S donor): 1) inhibited carbachol-induced contraction in muscle strips and isolated muscle cells that was independent of KATP channels, a known S-sulfhydration target of H2S; 2) induced S-sulfhydration of small G protein, RhoA leading to inhibition of RhoA and Rho kinase activities, a key pathway in the sustained smooth muscle contraction; and 3) inhibited PDE5 activity leading to augmentation NO-induced cGMP formation and muscle relaxation. Sodium nitroprusside (an NO donor) induced an increase in H2S production via PKG-dependent phosphorylation and activation of CSE. We conclude that smooth muscle cells selectively express CSE, and endogenous generation of H2S via activation of CSE inhibits muscle contraction and augments muscle relaxation. Inhibition of contraction is mediated via S-sulfhydration of RhoA and suppression of RhoA/Rho kinase pathway. Augmentation of relaxation is mediated via inhibition of PDE5 activity and stimulation of cGMP/PKG pathway, which in addition initiates generation of H2S via PKG-mediated phosphorylation and activation of CSE. The findings are important in providing the underlying mechanisms involved in the regulation of smooth muscle function by H2S and could offer insights for the development of therapeutic agents that may act on smooth muscle in the gut to treat motility disorders.

In the present study, we have examined the expression of protease-activated receptors (PARS) and characterized their signaling pathways in rabbit gastric muscle cells. Immunoblot analysis revealed expression of PARl and PAR2 but not PAR3 or PAR4 in smooth muscle. The PARl agonist TFLLR activated Gq, G12, and Gi3, but not Gil, Gi2, G13, Gs or Gz, whereas the PAR2 agonist SLIGRL activated Gq, G13, Gil, and Gi2, but not Gi3, G12, Gs, or Gz. Both PARl and PAR2 agonists stimulated PI hydrolysis and Rho kinase activity and inhibited cAMP formation. PAR1-stimulated PI hydrolysis was abolished in cells expressing Gαq minigene, but was not affected in cells expressing Gαi minigene or in cells treated with pertussis toxin (PTx). PAR2-stimulated PI hydrolysis was partially inhibited in cells expressing Gαq or Gαi minigene and in cells treated with PTx. PAR1- and PAR2-stimulated Rho kinase activity was abolished in cells expressing Gα12 or Gα13 minigene, respectively. Both PARl and PAR2 agonists induced a transient initial contraction that was selectively blocked by the inhibition of PI hydrolysis with U73122 and MLC kinase activity with ML-9. PAR1-induced sustained contraction was preferentially inhibited by the PKC inhibitor bisindolylmaleimide and to a minor extent by the Rho kinase inhibitor Y27632, whereas PAR2-induced sustained contraction was preferentially inhibited by Y27632. Activation of both PARl and PAR2 induced MLC20 phosphorylation, whereas phosphorylation of MYPTl and CPI-17 are receptor-specific: only PARl induced CPI-17 phosphorylation and only PAR2 induced MYPTl phosphorylation.Activation of PARl and PAR2 also induced IκBα degradation and NF-κB activation; the effects were abolished by the blockade of RhoA activity by Clostridium botulinum C3 exoenzyme suggesting NF-κB is downstream of RhoA. PAR1- and PAR2-stimulated Rho kinase activity was significantly augmented by the inhibitors of PKA (PKI), IKK2 (IKKIV), or NF-κB (MG132), and in cells expressing dominant negative mutants of IKK (IKK(K44A), IκBα (IκBα (S32A/S36A)), or phosphorylation-deficient RhoA (RhoA(S188A)). In addition, activation of PARl induced Gα12 phosphorylation, which was abolished by bisindolylmaleimide, suggests that phosphorylation was mediated by PKC derived from the activation of RhoA. Only PAR1-stimulated Rho kinase activity was significantly augmented by the PKC inhibitor. The effect of PKC inhibitor was additive to that of the PKA inhibitor.

Tonic arterial smooth muscle can produce strong contractions indefinitely by formation of slowly cycling crossbridges (latchbridges) that maintain force at a high energy economy. To fully understand the uniqueness of mechanisms regulating tonic arterial contraction, comparisons have been made to phasic visceral smooth muscles that do not sustain high forces. This study explored mechanisms of force maintenance in a phasic artery by comparing KCl-induced contractions in the tonic, femoral artery (FA) and its primary branch, the phasic saphenous artery (SA). KCl rapidly (5 N/m2) and [ca2+]i (250 nM) in FA and SA. By 10 min, [ca2+]i declined to 175 nM in both tissues but stress was sustained in FA (1.3 x 105N/m2) and reduced by 40% in SA (0.8 x l05 N/m2). Reduced tonic stress correlated with reduced myosin light chain (MLC) phosphorylation in SA (28% vs. 42% in FA). SA expressed more MLC phosphatase than FA, and permeabilized (β-escin) SA relaxed more rapidly than FA in the presence of MLC kinase blockade, suggesting that MLC phosphatase activity in SA was greater than that in FA. The reduction in MLC phosphorylation in SA was insufficient to account for reduced tonic force (latchbridge model), and SA expressed more "fast" myosin isoforms than did FA. Cytochalasin-D reduced force-maintenance more in FA than SA. These data support the hypothesis that strong force-maintenance is absent in SA because expressed motor proteins do not support latchbridge formation, and because actin polymerization is not stimulated.

Evidence shows the activation of neutrophils in the systemic vasculature of obese and preeclamptic women. In this study, I evaluated whether expression of neutrophil products, myeloperoxidase (MPO) and matrix metalloproteinase 8 (MMPS), was associated with neutrophil infiltration in systemic vascular tissue of obese and preeclamptic women. I tested my hypotheses by using immunohistochemical studies to look at the expression of MPO and MMP8 in the vasculature of obese and preeclamptic women. There was a significantly greater expression of MPO and MMP8 in the vasculature of preeclamptic women as compared to normal pregnant and normalnonpregnant women. The vasculature of obese women also had a significantly greater expression of MPO and MMP8 as compared to overweight and normal weight patients.These studies are the first to report that activated neutrophils in systemic vasculature are releasing MPO and MMP8. These findings also indicate that the vascular phenotype of obese and preeclamptic women is similar in that they both show an increased presence of MPO and MMP8 in the systemic vasculature as a result of neutrophil infiltration. This suggests that obese women are at increased risk for preeclampsia because their vasculature is already exposed to increased levels of MPO and MMP8, so when they become pregnant and experience further oxidative stress imposed by the placenta along with an increase in neutrophil number, they develop the clinical symptoms of preeclampsia.

It is well known that the overlap between the thick and thin filaments in striated muscle is responsible for the single active length-tension (L-T) curve. With the lack of visible striations, a sarcomeric unit has not been identified in smooth muscle. Though once thought to function like striated muscle via a sliding filament mechanism of contraction, recent studies on length-adaptation (L-adaptation) in airway smooth muscle (ASM), in which increased tension is generated with repeated contraction, have led to the hypothesis of a dynamic L-T curve in smooth muscle. Although more established in ASM, two studies have shown L-adaptation in vascular smooth muscle (VSM). In this project, the L-T curve over a 3-fold length range in rabbit femoral artery was investigated and the presence of more than one active and passive L-T curve was identified. The third of three repeated KCL-induced contractions at a single, shortened length resulted in L-adaptation in which the phasic and tonic phases of contraction demonstrated a 10-15% increase in active tension (Ta) relative to the first contraction. Experiments investigating possible mechanism(s) responsible for this phenomenon demonstrated that neither an increase in [Ca2+]i nor an increase in MLC20 phosphorylation was responsible for the increased tension. However, actin polymerization did appear to play a role in the L-adaptation of both phases of contraction. Thus directions for future research could include further study of actin polymerization in VSM that contributes to L-adaptation and may ultimately result in artery remodeling.