Resting and reactive blood pressure as predictors of ambulatory blood pressure in older hypertensive adults

Background. A review of the literature suggests that Hypertension (HTN) in older adults is associated with sympathetic stimulation that results in increasing blood pressure (BP) reactivity. If clinical assessment of BP captured sympathetic stimulation, it would be valuable for hypertension management. ^ Objectives. The study examined whether reactive change scores from a short BPR protocol, resting blood pressure (BP), or resting pulse pressure (PP) is a better predictor of 24 hour ambulatory BP and BP load in cardiac patients. ^ Method. The study used a single-group design, with both an experimental clinical component and an observational field component. Both components used repeated measurement methods. The study population consisted of 45 adult patients with a mean age of 64.6 ± 8.5 years who were diagnosed with cardiac disease and who were taking anti-hypertensive medication. Blood pressure reactivity was operationalized with a speech protocol. During the speech protocol, BP was measured with an automatic device (Dinamap 825XT) while subjects talked about their health and about their usual day. Twenty-four hour ambulatory BP measurement (Spacelabs 90207 monitor) followed the speech protocol. ^ Results. Resting SBP and resting PP were significant predictors of 24-hour SBP, and resting SBP was a significant predictor of SBP load. No predictors were significant of 24-hour DBP or DBP load. ^ Conclusions. Initial resting BP and PP may be used in clinical settings to assess hypertension management. Future studies are necessary to confirm the ability of resting BP to predict ABP and BP load in older, medicated hypertensives. ^

Attenuation of fibrosis in vitro and in vivo with SPARC siRNA.

INTRODUCTION: SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs. METHODS: In in vitro studies, skin fibroblasts obtained from a Tgfbr1 knock-in mouse (TBR1CA; Cre-ER) were transfected with SPARC siRNA. Gene and protein expressions of the Col1a2 and the Ctgf were examined by real-time RT-PCR and Western blotting, respectively. In in vivo studies, C57BL/6 mice were induced for skin and lung fibrosis by bleomycin and followed by SPARC siRNA treatment through subcutaneous injection and intratracheal instillation, respectively. The pathological changes of skin and lungs were assessed by hematoxylin and eosin and Masson's trichrome stains. The expression changes of collagen in the tissues were assessed by real-time RT-PCR and non-crosslinked fibrillar collagen content assays. RESULTS: SPARC siRNA significantly reduced gene and protein expression of collagen type 1 in fibroblasts obtained from the TBR1CA; Cre-ER mouse that was induced for constitutively active TGF-beta receptor I. Skin and lung fibrosis induced by bleomycin was markedly reduced by treatment with SPARC siRNA. The anti-fibrotic effect of SPARC siRNA in vivo was accompanied by an inhibition of Ctgf expression in these same tissues. CONCLUSIONS: Specific inhibition of SPARC effectively reduced fibrotic changes in vitro and in vivo. SPARC inhibition may represent a potential therapeutic approach to fibrotic diseases.

Neuronal and axonal degeneration in experimental spinal cord injury: in vivo proton magnetic resonance spectroscopy and histology.

Longitudinal in vivo proton magnetic resonance spectroscopy (1H-MRS) and immunohistochemistry were performed to investigate the tissue degeneration in traumatically injured rat spinal cord rostral and caudal to the lesion epicenter. On 1H-MRS significant decreases in N-acetyl aspartate (NAA) and total creatine (Cr) levels in the rostral, epicenter, and caudal segments were observed by 14 days, and levels remained depressed up to 56 days post-injury (PI). In contrast, the total choline (Cho) levels increased significantly in all three segments by 14 days PI, but recovered in the epicenter and caudal, but not the rostral region, at 56 days PI. Immunohistochemistry demonstrated neuronal cell death in the gray matter, and reactive astrocytes and axonal degeneration in the dorsal, lateral, and ventral white-matter columns. These results suggest delayed tissue degeneration in regions both rostrally and caudally from the epicenter in the injured spinal cord tissue. A rostral-caudal asymmetry in tissue recovery was seen both on MRI-observed hyperintense lesion volume and the Cho, but not NAA and Cr, levels at 56 days PI. These studies suggest that dynamic metabolic changes take place in regions away from the epicenter in injured spinal cord.

The Role of IL-6 in Adenosine-Mediated Pulmonary Fibrosis

Adenosine is a purinergic signaling molecule that regulates various aspects of inflammation and has been implicated in the pathogenesis of chronic lung diseases. Previous studies have demonstrated that adenosine up-regulates IL-6 production through the engagement of the A2B adenosine receptor in various cell types, including alveolar macrophages. IL-6 is elevated in mouse models and humans with chronic lung disease, suggesting a potential role in disease progression. Furthermore, chronic elevation of adenosine in the lungs of adenosine deaminase deficient (Ada-/-) mice leads to the development of pulmonary inflammation, alveolar destruction, and fibrosis, in conjunction with IL-6 elevation. Thus, it was hypothesized that IL-6 contributes to pulmonary inflammation and fibrosis in this model. To test this hypothesis, Ada/IL-6 double knockout mice (Ada/IL-6-/-) were generated to assess the consequences of genetically removing IL-6 on adenosine-dependent pulmonary injury. Ada/IL-6-/- mice exhibited a significant reduction in inflammation, alveolar destruction, and pulmonary fibrosis. Next, Ada-/- mice were treated systematically with IL-6 neutralizing antibodies to test the efficacy of blocking IL-6 on chronic lung disease. These treatments were associated with decreased pulmonary inflammation, alveolar destruction, and fibrosis. To determine the role of IL-6 in a second model of pulmonary fibrosis, wild type mice and IL-6-/- mice were subjected to intraperitoneal injections of bleomycin twice a week for four weeks. Results demonstrated that IL-6-/- mice developed reduced pulmonary fibrosis. To examine a therapeutic approach in this model, wild type mice exposed to bleomycin were treated with IL-6 neutralizing antibodies. Similar results were observed as with Ada-/- mice, namely diminished pulmonary inflammation and fibrosis. In both models, elevations in IL-6 were associated with increased phosphorylated STAT-3 in the nuclei of numerous cell types in the airways, including type II alveolar epithelial cells (AEC). Genetic removal and neutralization of IL-6 in both models was associated with decreased STAT-3 activation in type II AEC. The mechanism of activation in these cells that lack the membrane bound IL-6Ra suggests IL-6 trans-signaling may play a role in regulating fibrosis. Characterization of this mechanism demonstrated that the soluble IL-6Ra (sIL-6Ra) is upregulated in both models during chronic conditions. In vitro studies in MLE-12 alveolar epithelial cells confirmed that IL-6, in combination with the sIL-6Ra, activates STAT-3 and TWIST in association with enhancement of epithelial-to-mesenchymal transition, which can contribute to fibrosis. Similarly, patients with idiopathic pulmonary fibrosis demonstrated a similar pattern of increased IL-6 expression, STAT-3 activation, and sIL-6Ra increases. These findings demonstrate that adenosine-dependent elevations in IL-6 contribute to the development and progression of pulmonary inflammation and fibrosis. The implications from these studies are that adenosine and/or IL-6 neutralizing agents represent novel therapeutic targets for the treatment of pulmonary disorders where fibrosis is a detrimental component.

ROLE OF SOX9 IN UTERINE GLAND DEVELOPMENT AND DISEASE INITIATION

The female reproductive tract (FRT) develops midway through embryogenesis, and consists of oviducts, uterine horns, cervix and upper part of the vagina. The uterine horns are composed of an epithelial layer, luminal (LE) and glandular epithelium (GE), surrounded by a mesenchymal layer, the stroma and myometrium. Interestingly, in most mammals the GE forms after birth and it only becomes fully differentiated as the female reaches sexual maturity. Uterine glands (UG) are made up of GE and are present in all mammals. They secrete nutrients, cytokines and several other proteins, termed histotroph, that are necessary for embryo implantation and development. Experiments in ewes and mice have revealed that females who lack UGs are infertile mainly due to impaired implantation and early pregnancy loss, suggesting that UGs are essential for fertility. Fortunately for us, UGs develop after birth allowing us to peer into the genetic mechanism of tubulogenesis and branching morphogenesis; two processes that are disrupted in various adenocarcinomas (cancer derived from glands). We created 3D replicas of the epithelium lining the FRT using optical projection tomography and characterized UG development in mice using lineagetracing experiments. Our findings indicate that mouse UGs develop as simple tubular structures and later grow multiple secretory units that stem from the main duct. The main aim of this project was to study the role of SOX9 in the UGs. Preliminary studies revealed that Sox9 is mostly found in the nucleus of the GE. vii This observation led to the hypothesis that Sox9 plays a role in the formation and/or differentiation of the GE. To study the role of Sox9 in UGs differentiation, we conditionally knocked out and overexpressed Sox9 in both the LE and GE using the progesterone receptor (Pgr) promoter. Overexpressing Sox9 in the uterine epithelium, parts of the stroma, and myometrium led to formation of multiple cystic structures inside the endometrium. Histological analysis revealed that these structures appeared morphologically similar to structures present in histological tissue sections obtained from patients with endometrial polyps. We have accounted for the presence of simple and complex hyperplasia with atypia, metaplasia, thick-walled blood vessels, and stromal fibrosis; all “hallmarks” that indicate overexpressing Sox9 leads to development of a polyp-like morphology. Therefore, we can propose the use of Sox9-cOE mice to study development of endometrial cystic lesions and disease progression into hyperplastic lesions.

The role of adenosine in pulmonary angiogenesis

Angiogenesis is a feature of chronic lung diseases such as asthma and pulmonary fibrosis; however, the pathways controlling pathological angiogenesis during lung disease are not completely understood. Adenosine is a signaling nucleoside that accumulates as a result of tissue hypoxia and damage. Adenosine has been implicated in the exacerbation of chronic lung disease and in the regulation of angiogenesis; however, the relationship between these factors has not been investigated. The work presented in this dissertation utilized adenosine deaminase (ADA)-deficient mice to determine whether chronic elevations of adenosine in vivo result in pulmonary angiogenesis, and to identify factors that could potentially mediate this process. Results demonstrate that there is substantial angiogenesis in the tracheas of ADA-deficient mice in association with adenosine elevations. Replacement enzyme therapy with pegylated ADA resulted in a lowering of adenosine levels and reversal of tracheal angiogenesis, indicating that the increases in vessel number are dependent on adenosine elevations. Levels of the ELR+ angiogenic chemokine CXCL1 were found to be elevated in an adenosine-dependent manner in the lungs of ADA-deficient mice. Neutralization of CXCL1 and its putative receptor, CXCR2, in ADA-deficient lung lysates resulted in the inhibition of angiogenic activity suggesting that CXCL1 signaling through the CXCR2 receptor is responsible for mediating the observed increases in angiogenesis. Taken together, these findings suggest that adenosine plays an important role, via CXCL1, in the induction of pulmonary angiogenesis and may therefore represent an important therapeutic target for the treatment of pathological angiogenesis. ^

Deciphering the C-Type Lectin Receptor Signaling Pathway in Macrophages in Response to Candida albicans

Candida albicans causes opportunistic fungal infections in humans and is a significant cause of mortality and morbidity in immune-compromised individuals. Dectin-2, a C-type lectin receptor, is required for recognition of C. albicans by innate immune cells and is required for initiation of the anti-fungal immune response. We set out to identify components of the intracellular signaling cascade downstream of Dectin-2 activation in macrophages and to understand their importance in mediating the immune response to C. albicans in vivo. Using macrophages derived from Phospholipase-C-gamma 1 and 2 (PLCγ1and PLCγ2) knockout mice, we demonstrate that PLCγ2, but not PLCγ1, is required for activation of NF-κB and MAPK signaling pathways after C. albicans stimulation, resulting in impaired production of pro-inflammatory cytokines and reactive oxygen species. PLCγ2-deficient mice are highly susceptible to infections with C. albicans, indicating the importance of this pathway to the anti-fungal immune response. TAK1 and TRAF6 are critical nodes in NF-κB and MAPK activation downstream of immune surveillance and may be critical to the signaling cascade initiated by C-type lectin receptors in response to C. albicans. Macrophages derived from both TAK1 and TRAF6-deficient mice were unable to activate NF-κB and MAPK and consequently failed to produce inflammatory cytokines characteristic of the response to C. albicans. In this work we have identified PLCγ2, TAK1 and TRAF6 as components of a signaling cascade downstream of C. albicans recognition by C-type lectin receptors and as critical mediators of the anti-fungal immune response. A mechanistic understanding of the host immune response to C. albicans is important for the development of anti-fungal therapeutics and in understanding risk-factors determining susceptibility to C. albicans infection.

BIOCHEMICAL CHARACTERIZATION OF BINDING PARTNERS OF TWO HSP70 CO-CHAPERONES IN SACCHAROMYCES CEREVISIAE

Cells are exposed to a variety of environmental and physiological changes including temperature, pH and nutrient availability. These changes cause stress to cells, which results in protein misfolding and altered cellular protein homeostasis. How proteins fold into their three-dimensional functional structure is a fundamental biological process with important relevance to human health. Misfolded and aggregated proteins are linked to multiple neurodegenerative diseases, cardiovascular disease and cystic fibrosis. To combat proteotoxic stress, cells deploy an array of molecular chaperones that assist in the repair or removal of misfolded proteins. Hsp70, an evolutionarily conserved molecular chaperone, promotes protein folding and helps maintain them in a functional state. Requisite co-chaperones, including nucleotide exchange factors (NEFs) strictly regulate and serve to recruit Hsp70 to distinct cellular processes or locations. In yeast and human cells, three structurally non-related cytosolic NEFs are present: Sse1 (Hsp110), Fes1 (HspBP1) and Snl1 (Bag-1). Snl1 is unique among the cytosolic NEFs as it is localized at the ER membrane with its Hsp70 binding (BAG) domain exposed to the cytosol. I discovered that Snl1 distinctly interacts with assembled ribosomes and several lines of evidence indicate that this interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome-binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. In addition, I demonstrate ribosome association with the Snl1 homolog in the pathogenic fungus, Candida albicans and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. As a first step in determining specific domain architecture in fungal BAG proteins, I present the preliminary steps of protein purification and analysis of the minimal Hsp70 binding region in in both S.cerevisiae and C. albicans Snl1. Contrary to previous in vitro evidence which showed the Fes1 NEF to interact with both cytosolic Hsp70s, Ssa and Ssb, Fes1 is shown to interact specifically with Ssa when expressed under normal cellular conditions in S. cerevisiae. This is the first reported evidence of Hsp70 binding selectivity for a cytosolic NEF, and suggests a possible mechanism to achieve specificity in Hsp70-dependent functions. Taken together, the work presented in this dissertation highlights the striking divergence among Hsp70 co-chaperones in selecting binding partners, which may correlate with their specific roles in the cell.