CardioPulmonary Vascular Biology COBRE

The Cell Isolation & Organ Function Core provides a unique skill set and expertise to Rhode Island vascular biologists by providing quality assurance in isolation, characterization, and propagation of vascular derived cells and fibroblasts and cardiopulmonary organ function analyses. The centralization of the cell isolation and organ function measurements will help investigators minimize the variability in sample preparation thus providing uniformity in data acquisition throughout all COBRE Projects and for other RI vascular biologists.

Studies Overview

The overall objective of the CardioPulmonary Vascular Biology COBRE is to bring together a group of investigators in the fields of pulmonary and cardiovascular diseases in a unique trans-disciplinary approach to improve understanding of the pathogenesis and treatment of vascular pathobiology in lung and heart diseases. The Specific Aims of this proposal are:

  1. Unite a group of multidisciplinary investigators in studies of vascular injury and repair in the pathogenesis of lung and heart injury.
  2. Provide research support, protected time, and mentoring to enable junior investigators to attain independent research support in an “incubator” environment, coupled with a robust program for the nurturing of career development and attainment of additional research skills.
  3. Identify and support pilot investigators who will be funded to obtain preliminary data for research grants.
  4. Support junior and pilot investigators through mentoring and support from the Cores.
  5. Establish the basis for multidisciplinary program project grant(s) for research in vascular injury and repair.

cobre structureWe propose a Center that includes 5 junior investigators and 1-2 pilot investigators, supported by Administrative and Cell Isolation/Organ Function Cores. Junior and pilot investigators will be mentored by accomplished scientists with a strong focus on career development. A unique feature of the CardioPulmonary Vascular Biology COBRE is the melding under a common theme of vascular biology of research done by collaborating MDs, PhDs, and MD/PhDs. An outstanding group of scientists on the External Advisory Committee will provide advice, oversight, and critique. The Center will be administered by the Ocean State Research Institute, a non-profit research and education foundation located at the Providence VA Medical Center. The junior investigators and mentors are faculty at Alpert Medical School of Brown University, and they are employed by the Providence VAMC, Rhode Island Hospital, or Brown University. Generous and sustained institutional support has been provided for this COBRE. This innovative and multidisciplinary CardioPulmonary Vascular Biology COBRE will provide an environment that fosters creative and trans-disciplinary approaches to study of pathobiology of vascular components of lung and cardiovascular diseases. As the Center develops, the COBRE will be broadened to include other components of the vascular wall. By bringing together both basic and clinical scientists from cardiovascular and pulmonary backgrounds into a vascular biology center, this COBRE will enhance understanding of pathogenesis and treatment of vascular diseases and enhance research of these diseases, prevalent in RI.

COBRE Studies

Study 2 “Effects of Angiopoietins on shock/sepsis- induced acute lung injury”

PI: Joanne Lomas-Neira, PhD

COBRE ABSTRACT

Effects of Angiopoietins on shock/sepsis- induced acute lung injury

Acute Lung Injury (ALI) is a complication of trauma characterized by increased microvascular permeability, edema, inflammation, and neutrophil accumulation in the lung. Despite advances in supportive care, trauma patients who develop ALI, still face the risk of significant mortality. While neutrophils have been implicated as playing an important role in the pathogenesis of ALI, vascular endothelial cells (ECs), through their exposure/responsiveness to mediators present in the systemic environment, are also critical to the development of ALI. Using a murine model of hemorrhage (shock) in combination with a subsequent septic challenge, our laboratory has shown that neutrophil interactions with resident pulmonary cells are central to the development of trauma-induced ALI. EC activation, as in vessel remodeling and adhesion and migration of inflammatory cells, is an adaptive response, regulating vascular homeostasis and host defense, respectively. However, the loss of EC barrier function in ALI results in accumulation of fluid, proteins and inflammatory cells within the lung and in compromised gas exchange. This transition from adaptive activation to maladaptive/dysfunction occurs in association with unresolved inflammation. Under normal physiological conditions, regulated expression of two EC effector proteins, Angiopoietins (Ang)-1 & 2 mediates blood vessel formation, remodeling and EC interaction with inflammatory cells. Ang-1 & 2 bind to a tyrosine kinase receptor, Tie2, expressed on endothelial cells (EC). Ang-1 is produced by pericytes, smooth muscle cells, and fibroblasts and is found on the extracellular matrix. Ang-1/Tie2 binding promotes vessel integrity and downstream pro-survival and anti-inflammatory signaling. Ang-2 is stored preformed in EC Weibel-Palade bodies (storage vesicles) and is rapidly released upon EC activation. Unlike Ang-1, binding of Ang-2 plays a role in vascular remodeling and increased vessel permeability. The development of mice over-expressing or deficient in either Ang-1 or 2 has enabled a closer examination of the relationship between these two proteins and their regulation of EC function. Additionaly, it has been proposed that the relative expression of Ang-1 to Ang-2 determines EC responsiveness. In patients that develop ALI plasma Ang-2 levels are significantly elevated. Our recent findings show that Ang-2 is similarly elevated in lung tissue and plasma from mice following shock, in combination with a subsequent septic challenge. Importantly, neutropenic mice do not exhibit this increase. In addition, we have demonstrated that Ang-1:Ang-2 is significantly decreased in mouse lung tissue following shock/sepsis. This decreased ratio is also evident in bronchoalveolar lavage fluid from surgical and trauma ICU patients with ALI suggesting that the altered expression of these two proteins is either predictive or diagnostic of lung injury. However, the actual role Ang-2 plays in shock-induced priming for ALI and regulation of Ang-2’s release/expression has not been examined. To address this, the following central hypothesis is presented:
Angiopoietin-2 promotes loss of pulmonary endothelial barrier function in ALI resulting from the sequential insults of hemorrhagic shock and sepsis and this dysfunction is initiated by EC interaction with activated (shock primed) neutrophils.

Three aims are proposed to test this hypothesis. In Aim 1 we will determine the kinetics of change of Ang-1:Ang-2 in lung tissue and plasma following hemorrhagic shock and/or subsequent septic challenge and assess the role of Ang-1:Ang-2 in the development of ALI and mortality associated with multiple organ failure. This data will be used in Aim 2 to target Ang-1:Ang-2 at selected time points to determine the mechanisms by which Ang-2 changes receptor/adhesion molecule expression and loss of pulmonary endothelial barrier function in mice. In Aim 3 we will determine the degree to which direct EC/PMN interaction regulates the release of Ang-2 and the effects of changes in Ang-1:Ang-2. The data from the proposed study will begin to describe the mechanisms by which PMN-associated, Ang-2-mediated, EC activation leads to ALI. These results are anticipated to provide novel insights into the relationship between the role of angiopoietins in regulating vascular angiogenesis and PMN/EC interactions in the pathogenesis of ALI.

Ruhul Abid, MD

Study 3 “Ischemic heart disease (IHD)”

PI: Ruhul Abid, MD, PhD

COBRE ABSTRACT

Ischemic heart disease (IHD) or myocardial ischemia is a disease characterized by tissue hypoxia due to reduced blood supply to the heart muscle, usually caused by coronary artery disease. IHD is the leading cause of death and morbidity in the USA. Increase in coronary vessel diameter by vasodilatation (acute response), and increase in vessel density (delayed response) are two major defenses of myocardium from ischemic insults. While coronary vasodilatation is primarily dependent on endothelium-generated nitric oxide (NO), the increase in capillary density initially requires proliferation and migration of vascular endothelial cells (ECs). Increased levels of reactive oxygen species (ROS) are often observed in many cardiovascular diseases, including IHD, giving rise to the notion that ROS cause endothelial dysfunction. However, recent major interventional clinical trials using antioxidants (e.g. HOPE, ATBC), have largely produced negative results in reducing primary endpoints of cardiovascular death and morbidity. Reports from our lab demonstrated that reduced ROS levels inhibited signal transduction events that are essential for NO generation in the vascular endothelium and for coronary vasodilatation. Preliminary Results also showed that c-Src responds to changes in endothelial redox levels and promotes downstream PI3K-Akt signaling, which in turn activates eNOS and inhibits the growth inhibitory transcription factor, FOXO1, in coronary vascular ECs. This application will test a novel HYPOTHESIS that conditional increase in endothelium-specific-ROS will activate c-Src-PI3K-Akt-eNOS pathway and inhibit FOXO1, and thus, will result in coronary vasodilatation and increased vessel density in a myocardial ischemia model in vivo. Utilizing a newly developed binary transgenic mice that can induce conditional expression of Nox2 and 2-fold increase in ROS in vascular endothelium, we will determine whether EC-ROS activate c-Src-PI3K-Akt signaling, proliferation and migration of mouse heart ECs in vitro (Aim 1); whether EC-ROS induce PI3K-Akt-eNOS activation, NO synthesis and coronary vasodilatation (Aim 2); and whether EC-ROS increase vessel density in ischemic myocardium in an LAD ligation model in vivo (Aim 3).

Public Health Relevance:
The main objective of this proposal is to examine whether condtional increase (1.8±0.42-fold for 2 weeks) in EC-ROS improves coronary vascular tone and/or collateral vessel formation in the ischemic myocardium. The long-term goal is to develop redox-based novel therapeutic modalities that will ‘pre-condition’ coronary endothelium to improve coronary circulation in myocardial ischemia/infarction. Using novel binary transgenic mice (Tet-Nox2/VE-Cad-tTA) that, upon withdrawal of tetracycline, can induce 1.8±0.42-fold increase in EC-specific ROS in vivo, we propose the following Specific Aims:

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Study 4 “Regulation of Cardiac Fibroblast Function by MicroRNAs”

PI: Peng Zhang, MD, MS

COBRE ABSTRACT

Regulation of Cardiac Fibroblast Function by MicroRNAs

Fibrosis is an integral feature of the structural remodeling in the heart that occurs in response to a variety of cardiopulmonary diseases and can be a consequence of endothelial injury. It can impair ventricular function, increase the risk for arrhythmias and often leads to heart failure. Cardiac fibroblasts become activated in response to many forms of stress and play a critical role in fibrosis development. They are therefore important therapeutic targets, but therapies that specifically target fibroblasts are still at an early stage. Compared to traditional drug targets, microRNAs (miRNAs) offer novel mechanistic possibilities. Despite rapidly increasing insights into their roles in the heart, the understanding of miRNAs in physiological and pathophysiological processes is just emerging. Several miRNAs are known to regulate myocyte hypertrophy, excitation, contraction and survival; yet very little is known about their role in cardiac fibroblasts. The long-term goal of the miRNA research in my laboratory is to gain a better understanding of the functional role and mechanisms of action of miRNAs in cardiac fibroblasts under physiological and pathophysiological conditions. The central hypotheses of this study are (1) that miRNAs are dynamically regulated upon fibroblast activation and thereby regulate cardiac fibroblast function and (2) that miRNA manipulation in cardiac fibroblasts can be leveraged to prevent and/or reverse cardiac fibrosis development. The significance of the proposed study derives from mechanistic explorations that will provide comprehensive insights into miRNA alterations in cardiac fibroblasts in response to stress and to advance our understanding of the functional role and mechanisms of action of key miRNAs in cardiac fibroblasts, which may provide new opportunities for the treatment and prevention of cardiac fibrosis.

Jacob Moeller, BS, Research Assistant
Nedyalka (Nelly) Valkov, BS, MS, Brown University MPP Program PhD Student

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Study 5 “Sex and haemodynamics in Pulmonary Arterial Hypertension”

PI: Corey Ventetuolo, MD

COBRE ABSTRACT

Female sex is the best established risk factor for pulmonary arterial hypertension (PAH), implying estrogen has a harmful effect on the pulmonary vasculature. Although women are more likely to develop disease, right heart function (which determines prognosis) and survival appears to be better in women with PAH than in men. These paradoxical observations are as yet unexplained. We believe estrogen-mediated angiogenesis leads to changes in the pulmonary vascular bed but also increases in collateral blood flow in the right ventricle. The aim of this project are to correlate changes in angiogenesis markers (including endothelial progenitor cells and microparticles) with measures of pulmonary and right ventricular function during a normal menstrual cycle, which is characterized by fluctuation in estrogen, in PAH patients and in healthy women. We will study similar markers in post-menopausal women and capture hormonal exposures throughout a woman’s lifetime. Linking sex hormone pathways to angiogenesis and the right ventricle could open new avenues for research into the mechanisms of right heart failure, including promising new treatments that alter hormones in women (and men) with pulmonary vascular disease.

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Study 6 “Therapeutic Targeting of IL-1β-Based Mechanisms in Calcific Aortic Valve Disease.”

PI: Alan Morrison, MD

COBRE ABSTRACT

Calcific aortic valve disease (CAVD) is the most common cause of aortic stenosis in patients. The prevalence of aortic sclerosis, a precursor to stenosis, is approximately 25% of patients older than 65 years, and 10% of those patients will progress to severe, symptomatic stenosis. Pharmacotherapies aimed at slowing or reversing the disease process, including HMG-CoA reductase inhibitors (statins), have proven ineffective, and currently there is no medical therapy for CAVD. The current treatment paradigm targets symptomatic, severe stenosis (when mortality is high) with surgical aortic valve replacement, valvuloplasty, or transcatheter aortic valve replacement. The development of an effective medical treatment remains the major challenge for the field. My laboratory has developed a mouse model (Rac2-/-ApoE-/-) of accelerated inflammatory atherosclerotic calcification. The primary driver of progressive calcification is the cytokine, IL-1β, which promotes osteogenic transcription factor expression in vascular cells. The signaling mechanism responsible for increased IL-1β production in macrophages depends on a compensatory activation of Rac1. Preliminary data evaluating the aortic valve by histology and calcium-targeted imaging revealed evidence of aortic valve thickening and calcification, and treatment with the IL-1 receptor antagonist (IL-1ra) prevented progression of disease. The hypothesis is that macrophage Rac1-driven IL-1β expression is a critical mediator of the progression of CAVD. Using an established model of CAVD, we will identify macrophage Rac1 as a critical determinant of IL-1β expression and consequent progressive calcific aortic valve stenosis. Using diseased aortic valve tissue obtained at surgery from patients, we will define the relevance of this Rac-dependent IL-1β expression to human CAVD. Because inhibition of IL-1β, using IL-1ra or inhibitory monoclonal antibody, is approved for treatment of certain inflammatory diseases, we see this pilot study as critical to highlighting the mechanistic basis for inhibition of IL-1β as a novel preventive treatment strategy for patients afflicted with CAVD. Moreover, this study will establish the potential viability of Rac1-targeted inhibitors in treating this disease process.