Vascular Responses to Pathogens

Vascular Responses to Pathogens

Edited by

Felicity N.E. Gavins

Department of Molecular and Cellular Physiology, Center for Cardiovascular Diseases and Sciences, and Department of Neurology, LSU Health Sciences Center, Shreveport, LA, USA

Karen Y. Stokes

Department of Molecular and Cellular Physiology, Center for Cardiovascular Diseases and Sciences, and Center for Molecular and Tumor Virology, LSU Health Sciences Center, Shreveport, LA, USA

Table of Contents

Cover

Title page

Copyright

List of Contributors

Overview

Chapter 1: Sepsis

Abstract

Introduction

The microcirculation in sepsis

The endothelium

Nitric oxide and sepsis

Arterioles

Capillaries

Venules

Permeability

Blood cells

Platelets in sepsis

Thrombosis and coagulation in sepsis

Models

Genetics and sepsis

Treatments for sepsis

Conclusions

Acknowledgment

Chapter 2: Neutrophil-Mediated Vascular Host Defense

Abstract

Introduction

Bloodstream infections

Conventional vascular host defense

Neutrophil extracellular trap mediated vascular immunity

Immunothrombosis

NET-mediated tissue damage

Conclusions

Chapter 3: Platelets as Mediators of the Vascular Response to Infection

Abstract

Introduction

Platelet and hemostasis

Platelets and the response to infection

Platelet–bacteria interactions

Direct binding

Indirect binding

Shear stress

Secreted products

Platelet activation

Platelets and sepsis

Conclusions

Chapter 4: Mechanisms of Bacterial Interaction with Cells of the Blood–Cerebrospinal Fluid Barrier

Abstract

Introduction

Bacterial meningitis

Blood–brain barrier/blood–cerebrospinal fluid barrier

Bacterial ligand–receptor interactions

Signal-transduction mechanisms

Analysis of the host cell transcriptome in response to bacterial infection

Summary

Acknowledgments

Chapter 5: Vascular Responses to Chlamydia pneumoniae Infection

Abstract

Introduction

Direct interaction with vascular cells

Systemic responses to infection and the impact on the vasculature

Conclusions

Chapter 6: Bartonella Species and Vascular Pathology

Abstract

Introduction

Bartonellosis: the historical perspective

Pathogenesis of Bartonella-induced vasculoproliferative disease

Clinical manifestations of bartonellosis and Bartonella-associated vasoproliferative disease

Therapeutic strategies

Conclusions

Future research directions

Chapter 7: Periodontal Innate Immune Mechanisms Relevant to Atherosclerosis

Abstract

Introduction

Periodontal disease’s impact on the immune response

Homotolerance

Role of periodontal disease and its related bacteria in atherosclerosis

Immune response: role of NOD in infection-inflammation and atherosclerosis

Role of NOD2 in P. gingivalis detection within the endothelial and its association with atherosclerosis

Discussion

Acknowledgment

Chapter 8: Helicobacter pylori

Abstract

Introduction

The contribution of H. pylori to cardiovascular risk due to overall pathogen burden

Epidemiological studies of H. pylori in vascular disease

The role of H. pylori virulence factors in vascular disease risk

Potential mechanisms

Concluding remarks and perspective

Acknowledgment

Chapter 9: Endothelial Activation and Injury: The Mechanisms of Rickettsial Vasculiti

Abstract

Rickettsial diseases and host cell tropism in vivo

Rickettsial interactions with vascular endothelium: adhesion and invasion

Rickettsia infection of cultured human endothelial cells in vitro

Endothelial cell activation in response to Rickettsia infection in vitro

Endothelial cell activation consequent to Rickettsia infection in vivo

Conclusions

Acknowledgments

Chapter 10: Herpesviruses

Abstract

Introduction

Epidemiology

Viral dissemination

Association of herpesviruses with vascular diseases

Herpesviruses may alter nitric oxide and cause oxidative stress

Herpesviruses induce inflammatory and immune responses

Platelets and thrombosis

Angiogenesis

Herpesviruses may alter other cardiovascular risk factors

Conclusions

Chapter 11: HIV-1 Infection, Antiretroviral Therapies, and HIV-Associated Atherosclerosis

Abstract

Introduction

Epidemiological support for HIV-associated atherosclerosis

Mechanisms of vascular damage

Conclusions

Chapter 12: Viral Myocarditis and Dilated Cardiomyopathy: Mechanisms of Cardiac Injury, Inflammation, and Fibrosis

Abstract

Myocarditis and dilated cardiomyopathy

Stages in the viral myocarditis/DCM disease process

Conclusions

Acknowledgments

Chapter 13: Viruses Responsible for Hemorrhagic Fevers

Abstract

Introduction

Viral factors mediating direct effects on vascular endothelium

Indirect mechanisms of endothelial activation and their effects on vascular barrier and plasma leakage

Vascular endothelium and hemostasis: thrombocytopenia and coagulopathy during VHD

Activation of the coagulation system during VHD

Conclusions

Abbreviations

Chapter 14: Trypanosoma cruzi and Chagas Disease: Innate Immunity, ROS, and Cardiovascular System

Abstract

Life cycle and epidemiology of Trypanosoma cruzi infection

Clinical Chagas disease

Pathomechanisms of Chagas disease

Summary and future perspectives

Acknowledgments

Chapter 15: Endothelial Cells as Targets of the Intravascular Parasitic Disease Schistosomiasis

Abstract

Introduction to schistosomiasis

The intravascular parasite life cycle: dangerous liaisons

Clinical manifestations and immunological responses in schistosomiasis

Lung vascular responses during schistosomiasis

Portal and mesenteric vascular system alterations during schistosomiasis

Ectopic schistosomiasis

Conclusions

Chapter 16: Vascular Responses in Human Lymphatic Filariasis

Abstract

Introduction

Clinical manifestations

Vascular responses in lymphatic filariasis

Conclusions

Acknowledgment

Conflict of Interest Disclosure

Chapter 17: The Treatment of Sepsis: From Failed Therapies to New Possibilities

Abstract

Introduction

What is sepsis?

Problem with the definition

Moving from bench-side research to clinical use

Forty years of trials (and error)

Current therapies in sepsis

Conclusions (a primer for future preclinical studies into sepsis)

Subject Index

Customer survey

Copyright

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List of Contributors

Jacob Al-Hashemi,     Center for Anti-inflammatory Therapeutics, School of Dental Medicine, Boston University, Boston, MA, USA

Salomon Amar,     Center for Anti-inflammatory Therapeutics, School of Dental Medicine, Boston University, Boston, MA, USA

Subash Babu,     NIH-NIRT-ICER, Chennai, India

Edward B. Breitschwerdt,     Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA

Jerry L. Brunson,     Department of Molecular and Cellular Physiology, Center for Cardiovascular Diseases and Sciences, LSU Health Sciences Center, Shreveport, LA, USA

Iwona Buskiewicz,     Department of Pathology, University of Vermont, Colchester, VT, USA

Lee Ann Campbell,     Departments of Environmental and Occupational Health Sciences and Global Health, School of Public Health, University of Washington, Seattle, WA, USA

Han-Oh Chung,     Department of Medicine, Division of Critical Care, McMaster University, Hamilton, ON, Canada

Vivian Vasconcelos Costa

Laboratório de Interação Microrganismo-Hospedeiro, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte

Imunofarmacologia, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

Interdisciplinary Research Group in Infectious Diseases, Singapore-MIT Alliance for Research and Technology, Singapore

Dermot Cox,     RCSI Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin, Ireland

Danielle da Gloria de Souza

Laboratório de Interação Microrganismo-Hospedeiro, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte

Imunofarmacologia, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

Mahalia S. Desruisseaux,     Departments of Pathology and Medicine, Albert Einstein College of Medicine, Bronx, NY, USA

Kelly S. Doran,     Department of Biology and Center for Microbial Sciences, San Diego State University, San Diego, CA, USA

Tammy R. Dugas,     Department of Comparative Biomedical Sciences, LSU School of Veterinary Medicine, Baton Rouge, LA, USA

DeLisa Fairweather,     Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA

Alison E. Fox-Robichaud,     Department of Medicine, Division of Critical Care, McMaster University, Hamilton, ON, Canada

Nisha J. Garg,     Department of Microbiology and Immunology; Department of Pathology, and Institute for Human Infections and Immunity and the Center for Tropical Diseases, University of Texas Medical Branch, Galveston, TX, USA

Felicity N.E. Gavins,     Department of Molecular and Cellular Physiology, Center for Cardiovascular Diseases and Sciences, and Department of Neurology, LSU Health Sciences Center, Shreveport, LA, USA

Mitzi C. Glover,     Department of Cell Biology & Anatomy, LSU Health Sciences Center, New Orleans, LA, USA

Sally Huber,     Department of Pathology, University of Vermont, Colchester, VT, USA

Mikhail V. Khoretonenko,     Department of Biology, Lakeland Community College, Kirtland, OH, USA

Jung Hwan Kim,     Snyder Institute for Chronic Diseases, University of Calgary; Department of Critical Care Medicine, Faculty of Medicine, University of Calgary, Calgary, Canada

Dorsey L. Kordick,     Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA

Fabiana S. Machado,     Infectious Diseases and Tropical Medicine/Interdisciplinary Laboratory of Medical Investigation, Faculty of Medicine and the Department of Biochemistry and Immunology, Institute of Biological Science, Federal University of Minas Gerais, Belo Horizonte, Brazil

Claudia Lucia Martins Silva,     Pharmacology and Inflammation Research Program, Institute of Biomedical Sciences, Federal University of Rio de Janeiro (UFRJ), CCS, Rio de Janeiro, Brazil

Mauro Martins Teixeira,     Imunofarmacologia, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

Hema P. Narra,     Department of Pathology, University of Texas Medical Branch, University Boulevard, Galveston, TX, USA

Thomas B. Nutman,     Helminth Immunology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA

Carlos Robello,     Department of Biochemistry, School of Medicine, and Unit of Molecular Biology, Institut Pasteur de Montevideo, Montevideo, Uruguay

Bram Rochwerg,     Department of Medicine, Division of Critical Care, McMaster University, Hamilton, ON, Canada

Michael E. Rosenfeld,     Departments of Environmental and Occupational Health Sciences and Global Health, School of Public Health, University of Washington, Seattle, WA, USA

Abha Sahni,     Department of Pathology, University of Texas Medical Branch, University Boulevard, Galveston, TX, USA

Sanjeev K. Sahni,     Department of Pathology, University of Texas Medical Branch, University Boulevard, Galveston, TX, USA

Alexandra Schubert-Unkmeir,     Institute of Hygiene and Microbiology, University of Wuerzburg, Wuerzburg, Germany

Karen Y. Stokes,     Department of Molecular and Cellular Physiology, Center for Cardiovascular Diseases and Sciences; Center for Molecular and Tumor Virology, LSU Health Sciences Center, Shreveport, LA, USA

Herbert B. Tanowitz,     Departments of Pathology and Medicine, Albert Einstein College of Medicine, Bronx, NY, USA

Traci L. Testerman,     Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, SC, USA

David H. Walker,     Department of Pathology, University of Texas Medical Branch, University Boulevard, Galveston, TX, USA

Jian-jun Wen,     Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA

Bryan G. Yipp,     Snyder Institute for Chronic Diseases, University of Calgary; Department of Critical Care Medicine, Faculty of Medicine, University of Calgary, Calgary, Canada

Overview

Felicity N.E. Gavins

Karen Y. Stokes

It is now a century since infectious diseases were first associated with atherosclerotic lesions. Since then, several bacteria, viruses, and parasites have been implicated in different inflammatory diseases of the vasculature, including cardiovascular disease, autoimmune disease, cancers, and sickle cell disease. While for some pathogens, it has been relatively easy to show causative or contributing roles through epidemiological studies, for others, such as cytomegalovirus, their high prevalence in the population has made this more difficult. Nonetheless, evidence is building to suggest that bacteria, viruses, and parasites can target vessel walls and different blood cell populations and immune responses to induce or worsen vascular pathologies. This book aims to cover the multifaceted consequences on the vasculature of different pathogens by gathering leading experts from around the world who can provide evidence encompassing micro- and macrovascular responses to bacteria, parasites, and viruses.

The concept for this book originally came from a conference symposium we developed. We were somewhat surprised that despite the growing evidence of the roles for different pathogens in vascular diseases, there had not been a concerted effort to bring the evidence together in one book. This pushed us to expand upon our initial idea, and to incorporate the multiple facets of infectious diseases in this book. The chapters within provide basic scientists and clinicians with epidemiological evidence implicating various pathogens in vascular dysfunction (which in some cases is still controversial) as well as mechanistic insights into how the pathogens are acting. As will be seen with many of the pathogens, it is clear that they promote responses in different cell types important for vascular disease as well as in animal models, but, similar to many other diseases, translation to the human population has proven more difficult. The variability in measurements, study populations, geographical differences, and so on, can in part explain this. We hope that you, the readers, will find the information useful for your research or clinical practice, with a view to moving the field forward toward treating or preventing the pathogen components of vascular diseases.

Finally, we would like to thank Shannon and Stacey at Elsevier for their constant encouragement and help throughout this process. In addition, we are immensely grateful to all of the authors and reviewers who made the book possible. We hope you enjoy the book!

Chapter 1

Sepsis

Felicity N.E. Gavins    Department of Molecular and Cellular Physiology, Center for Cardiovascular Diseases and Sciences, and Department of Neurology, LSU Health Sciences Center, Shreveport, LA, USA

Abstract

Sepsis triggers both cellular and humoral aspects of the inflammatory immune response. The assortment of pathogenic factors (such as the bacterial fragment, lipopolysaccharide (LPS)) triggers every component of the microcirculation, including (but not limited to): endothelial cells, platelets, leukocytes (e.g., neutrophils and monocytes/macrophages), erythrocytes, the coagulation cascade, and smooth muscle cells. It is agreed that it is not the body’s response to the infection itself that causes the problems associated with sepsis, but rather the inflammatory response that is evoked by the infection. This chapter will provide an overview of the microvascular dysfunction induced by sepsis (involving arterioles, capillaries, and venules), current concepts, and potential therapeutic strategies.

Keywords

microcirculation

tissue oxygenation

nitric oxide

adhesion molecules

leukocyte recruitment

vascular permeability

Introduction

Sepsis is a major cause of death and disability worldwide. In North America, at the time of going to press, over one million cases of sepsis occur annually, with 40% leading to severe sepsis (3% of those with severe sepsis experience septic shock) and 300,000 deaths. This is a huge economic burden, with over 45% of the total intensive care unit costs being spent on treatment.¹,² A number of reasons are recognized for the growing incidence of sepsis, such as increasing antibiotic resistance, an increase in the aged population, and an increase in the use of invasive procedures. Other factors that predispose people to septic shock include cancer, immunodeficiency, chronic organ failure, and genetic factors.³

The discovery of sepsis dates back to 1879–1880, when Louis Pasteur showed for the first time that bacteria were present in blood from patients with puerperal septicemia. However, the consensus to define sepsis clinically is relatively recent. Sepsis is defined as an infection with evidence of systemic inflammation, consisting of two or more of the following: increased or decreased temperature or leukocyte count, tachycardia, and rapid breathing. (The history of sepsis and the clinical definitions are covered in Chapter 13.)

Sepsis is also defined as a systemic inflammatory response syndrome, due to its ability to affect organ systems remote from the site of infection. Disseminated intravascular coagulation (DIC), which is an acquired syndrome characterized by the activation of intravascular coagulation, is involved in both the pathogenesis of sepsis and the development of multiple organ dysfunction syndrome (MODS). DIC culminates in intravascular fibrin formation and deposition in the microvasculature,⁴,⁵ which leads to obstruction of blood flow, and ultimately progressive organ dysfunction. DIC can frequently be found in 25–50% of patients with sepsis, and appears to be a strong predictor of mortality.⁶

Sepsis-related cardiovascular changes are complex and include vasodilation, hypervolemia, and myocardial depression.⁷ Cerebrovascular complications also occur, and brain dysfunction is often one of the first clinical signs in sepsis and may manifest as sepsis-associated delirium in up to 70% of patients.⁸ Sepsis is also characterized by a number of circulatory disturbances, including decreased systemic vascular resistance, hypotension, hypoperfusion, impaired oxygen utilization, lactic acidosis, and impaired blood flow in the microcirculation and microvascular thrombosis.⁹

The microcirculation in sepsis

The microcirculation (defined as blood vessels with diameter <100 mm) is an organ of the cardiovascular system that supplies tissues and cells with oxygen and nutrients, and removes waste products, all of which are needed for survival. The microcirculation plays a dominant role in sepsis, and is a major contributing factor to MOD, which is itself predictive of mortality in sepsis¹⁰,¹¹ (Fig. 1.1). Each compartment of the microcirculatory unit plays a role, that is, the endothelium, arterioles, capillaries, venules, and blood cells. Furthermore, studies have shown that septic patients who have improved microcirculatory flow during the resuscitation phase of therapy, have reduced MOD.¹²

Figure 1.1   A schematic of some of the platelet–neutrophil–endothelial interactions that occur during sepsis.

During sepsis, CD40L is expressed and shed by platelets and interacts with CD40 on neutrophils. Adhesion molecule P-selectin is released from neutrophil α-granules, which binds to PSGL1 on platelets, and neutrophils themselves interact with cell adhesion molecules (CAMs) on the endothelial surface. These interactions cause the activation of neutrophils, which release reactive oxygen species (ROS) that destroy bacteria and promote further recruitment of neutrophils to sites of inflammation. Activation of leukocyte function-associated antigen 1 (LFA-1) on platelets also targets bacteria, by triggering the release of neutrophil extracellular traps (NETs), primarily composed of DNA and extracellular fibers, which promote thrombus formation and trap free bacteria.

The endothelium

The microcirculation is lined with a continuous single-(endothelial) cell lining, which is covered with a mechanotransducer membrane (the glycocalyx, which extends luminally from several nanometers to 3 μm) that enables the endothelial cells to sense changes in blood flow and plays a major role in regulating circulatory fluid homeostasis and vessel integrity.¹³,¹⁴ The endothelium is an intricate organ comprised of 10¹² cells, covering 4000–7000 m² in an adult. Under normal situations, the endothelium is unactivated, and as such is an antiadhesive and antithrombotic surface (through its production of thrombomodulin, which activates protein C; tissue factor pathway inhibitor (TFPI); heparin, which potentiates the action of antithrombin III; and tissue plasminogen activator (TPa)). It regulates blood flow and controls the contractility of vascular smooth muscle cells mainly by the synthesis and release of nitric oxide (NO).¹⁵,¹⁶ In inflammatory situations, the protective effect of NO decreases, due to the production of superoxide (the source of which is likely to be NADPH oxidase because it is colocalized with endothelial-derived NO synthase (eNOS) in subcellular compartments within endothelial cells).¹⁷,¹⁸

In sepsis, endothelial cells are exposed to a variety of blood-borne endotoxins (e.g., Escherichia coli lipopolysaccharide (LPS)) and inflammatory cytokines (e.g., interferon-γ (IFNγ)).¹⁷ The glycocalyx is shed and syndecan-1 (a glycocalyx marker) is increased in serum samples from septic patients, along with the inflammatory markers intracellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), and interleukin 6 (IL-6),¹⁹ which lead to cellular adhesion. Coupled to this, gap junctions are disturbed (allowing for adhesion and emigration of leukocytes,¹⁹,²⁰ increased endothelial permeability (which in turn leads to a loss of blood volume and progression of septic shock²¹–²³ and a heightened inflammatory response), ROS, reactive nitrogen species (RNS), and other cytokines (e.g., IL-1β and tumor necrosis factor-α (TNF-α), which also activates inducible nitric oxide synthase (iNOS)) are all released, and tissue hypoxia develops (because the diffusion distance for oxygen increases between blood and tissue cells). Thus, these events propagate the activated endothelium to assume a proadhesive and prothrombotic phenotype, and thereby contributing to the onset of MODS and death.

Nitric oxide and sepsis

NO is an important cellular signaling molecule, produced by the endothelium, and plays a fundamental role in maintaining the flow in microvessels by mediating vascular tone, leukocyte adhesion, platelet aggregation, microthrombi formation, and microvascular permeability.²⁴–³² Due to its importance in all these physiological processes, abnormalities in the NO system is one of the key mechanisms associated with severe sepsis and septic shock. NO is generated from l-arginine by the enzyme NOS.³³ There are three different NOS isoenzymes: two are produced in small quantities, constitutively expressed and is calcium dependent (endothelial and neuronal; eNOS and nNOS, respectively) and one that is produced in large quantities, and is a calcium-independent inducible isoform, iNOS.³³ Endotoxins and cytokines activate iNOS, which leads to local and general vasodilatation,⁸,³⁴,³⁵ and as such, is important in host defense. However, the variation of iNOS and the consumption of NO by ROS may lead to the known pathologic shunting of oxygenated blood from susceptible tissues, which characterizes the heterogeneous tissue perfusion of sepsis.³,³⁶,³⁷ Interestingly, pharmacologic inhibition of iNOS activity restores resistance vessel reactivity to near normal (Fig. 1.1);³³ however, one randomized controlled trial of nonspecific iNOS blockade was stopped due to increased mortality in the iNOS blockade group.³,³⁷ Thus, NO may actually be an adaptive response that is an attempt to restore blood flow at the level of capillaries.³

Arterioles

The arterial endothelium plays a pivotal role, regulating the vessel resistance and fluid homeostasis between blood and the interstitial space, under physiological conditions.³⁸ Arterioles are classed as resistance vessels because of their ability to dilate and constrict blood flow over relatively long distances (order of 1 mm) in order to regulate microvascular blood flow. The blood flow in these vessels is under the influence of intrinsic and extrinsic factors. In sepsis (or endotoxemia), vasoconstrictors and vasodilators diminish the blood flow in many different vascular beds.³³ It also interferes with pressure-flow regulation (i.e., the ability of an organ to maintain its blood flow) in the liver³⁹ but not in the brain.³³,⁴⁰,⁴¹ Of significance, at the microvascular level, sepsis causes constriction in larger (>80 mm diameter) arterioles, and dilation in smaller terminal arterioles (<80 mm diameter). This constriction of large-inflow arterioles reduces the overall blood volume delivered to capillaries and may ultimately contribute to metabolic acidosis.³³ Morphological alterations or arteriolar endothelial damage can result in sepsis-induced arteriolar hyporesponsiveness. Endotoxemia can cause endothelial cell swelling and pseudopod formation⁴² or even to denudation of the endothelial lining.⁴³

In summary, sepsis results in arteriolar dysfunction, causing an inability of these vessels to respond to intrinsic, and more importantly, extrinsic stimuli, which regulate blood flow and oxygen delivery.³³

Capillaries

Despite the capillaries being the smallest of the blood vessels (with diameters of 5–10 μm) in the microcirculation, they play an extremely important role of providing oxygen delivery and nutrients to tissues, and the removal of carbon dioxide and waste products. In sepsis, the function of the capillaries is compromised, and are often characterized by decreased functional capillary density, increased perfusion heterogeneity, and increased proportion of stopped and intermittently perfused capillaries. As blood flow increases through the capillaries, due to decreased oxygen delivery or an increase in oxygen demand, the capillary bed becomes more homogeneous, resulting in a more uniform delivery of oxygen to the tissue.³³,⁴⁴,⁴⁵ This lack of oxygen delivery to the cell, and in particular to mitochondria within the cell, instigates the waning of mitochondrial oxidative phosphorylation, bringing about anaerobic glycolysis and the production of cellular lactate (as such lactate is often used as an indicator of tissue hypoperfusion). These effects result in the eventual diffusion into the blood during prolonged cell ischemia.⁴⁶

Sepsis is associated with a decreased tissue capillary density, and an increase in the number of plugged capillaries (i.e., impaired capillary blood flow, or no reflow), which was first verified in striated muscle from septic rats,³⁶ using the technique of intravital microscopy.⁴⁷ In the brain, increased leukocyte transmigration and an increase in the perivascular edema lead to a decrease in blood flow in the cerebral capillaries. It has also been shown, using knockout mice (in both gp91phox knockout (a subunit of NADPH oxidase), and NOS knockout animals) that NADPH oxidase-ROS plays a greater role in capillary impairment versus NO.⁴⁴ Intravital microscopy studies have also indicated that capillaries can respond to vasoactive agents by signaling upstream arterioles to alter their vascular tone,⁴⁸ which is likely due to the electrotonic spread of the signal along the endothelium.³³ However, these events all play a role in the decreased oxygen delivery that is common in sepsis, as well as redistribution of blood flow, which leads to regional hypoxia⁴⁹ and organ failure.³,³³ Clinical trials have demonstrated these effects and have demonstrated that microvascular perfusion improves in survivors from septic shock but not in nonsurvivors,³ even when shock resolves.³

To summarize, sepsis reduces the number of perfused capillaries, which leads to a decrease in oxygen delivery to the mitochondria and increased hypoxia and ischemic injury.

Venules

Twenty-five percent of the total blood volume is contained in vessels of 7–50 μm diameter, termed venules (veins contain approximately 70%). Sepsis and endotoxemia instigate inflammation, and venules are the primary site for this inflammation and the body’s inflammatory response to take place. It is characterized by an infiltration of leukocytes, which is achieved by a complex interaction between selectins and integrins in response to chemoattractants such as complement factors, leukotriene B4 (LTB4), and platelet-activating factor. This inflammatory process involves a number of intricate and complex steps, including leukocyte capture (or tethering), slow rolling, adhesion strengthening and spreading, intravascular crawling, and transmigration (either via a transcellular (through endothelial cells) or a paracellular (between endothelial cells) route) into the site of tissue injury⁵⁰ (see section entitled The Inflammatory Response in Sepsis for more details).

In summary, sepsis causes an inflammatory response in venules, as demonstrated by exaggerated leukocyte infiltration, endothelial swelling, and protein leakage. However, despite the detrimental effects of the inflammatory response, it is crucial that the inflammatory response is allowed to happen, as it also upregulates anti-inflammatory mechanisms, which in turn will help aid the resolution.

Permeability

One of the five cardinal signs of inflammation is tumor or swelling (the others being rubor (redness), calor (increased heat), dolor (pain), and functio laesa (loss of function)). Sepsis and endotoxemia increase microvascular permeability, which is dependent³³ and independent³³ on leukocyte emigration. The choice of dependence or independence could simply be due to concentration of inflammatory markers produced that reach such a level as to be able to cause endothelial cell retraction, without the need of leukocyte emigration. Indeed, superfusion of the hamster cheek pouch with LPS increased macromolecular leakage without any changes in cellular interactions, as observed using intravital microscopy.⁵¹

Blood cells

During sepsis, the immune response is deemed to be depressed, as shown by the hyporeactivity of lymphocytes and decreased numbers (due to increased apoptosis) in the circulation and tissue of patients with sepsis.⁵² Macrophages and neutrophils are two innate immune cell types intimately associated with the excessive inflammatory response characterizing severe sepsis. Sepsis and endotoxemia induce key changes in monocytes and macrophages via cell surface makers such as chemokine CXC receptor 2 (CXCR2), TNF receptor p50 and p75, C5a receptor, toll-like receptors 2 and 4, and CD14.³,⁵³,⁵⁴

Neutrophils are one of the most abundant cells of the immune system, and are well suited to be able to fight infection, as they are armed with a variety of antimicrobial substances, proteases and ROS. The cytoplasmic granules in the neutrophil store the majority of these molecules, which enables large amounts to be stored, and are readily available as and when required.⁵⁵ This granular storage system also serves to protect the neutrophil itself from many of these nonspecific and toxic substances, which could damage the neutrophil extracellular matrix and surrounding cells.

The Inflammatory Response in Sepsis

For many years, the inflammatory response was thought to subside by the passive action of the dissipation of proinflammatory mediators; however, it is now known that resolving phase of inflammation is not a passive process, but actively switches off via the biosynthesis of endogenous anti-inflammatory mediators⁵⁶,⁵⁷ including the glucocorticoid-induced calcium and phospholipid-binding protein, annexin A1 (AnxA1⁵⁸), lipoxins, resolvins, protectins, and maresin families (the latter four are collectively called specialized proresolving mediators (SPMs)).⁵⁷

Neutrophil apoptosis is not only central to the prevention of inflammation, but it also signals the resolution of inflammation. Under normal inflammatory conditions, apoptotic neutrophils are engulfed by macrophages, which provide a strong signal for the resolution of inflammation. However, in sepsis, neutrophil apoptosis is inhibited. This effect is thought to be due to the activity of circulating factors, including LPS, lipoteichoic acid, and proinflammatory cytokines.⁵⁹–⁶³ Alongside this, sepsis also produces antiapoptotic factors, which further extends the neutrophil lifespan (typically under basal conditions the average circulatory lifespan of neutrophils is up to 12.5 h for murine cells and 5.4 days for human neutrophils),⁶⁴ increasing the probability of these cells to bring about extracellular damage through their uncontrolled release of oxygen radicals and proteolytic enzymes, such as myeloperoxidase (MPO).⁶⁵

The recruitment of leukocytes to injured or infected tissue is a central element to the inflammatory response and in numerous studies has been demonstrated to act as a rate-limiting step.⁶⁶ Therefore, targeting this process may provide a key point of intervention in the inflammatory cascade, and understanding the mechanisms governing leukocyte recruitment is vital in developing effective therapies.

The initial capture and rolling of leukocytes is mediated by the interactions of the selectins (P-selectin, also known as CD62P, granule membrane protein 140 (GMP-140), and platelet activation-dependent granule to external membrane protein (PADGEM), L-selectin (CD62L), and E-selectin, also known as CD62E, endothelial-leukocyte adhesion molecule 1 (ELAM-1), or Leukocyte endothelial cell adhesion molecule 2 (LECAM2), named in relation to the cell type on which they were originally identified (platelet, leukocyte, and endothelium)) with their ligands (typically P-selectin glycoprotein ligand 1 (PSGL1), E-selectin ligand, glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1), mucosal vascular addressin cell adhesion molecule 1 (MadCAM-1), and hematopoietic progenitor cell antigen CD34 (CD34)). P-selectin is stored in the Weibel–Palade bodies of endothelial cells and neutrophil α-granules, and is the earliest adhesion molecule to be upregulated (within 3–10 min) on the luminal–endothelial surface in response to proinflammatory signals such as LPS. E-selectin on the other hand is not stored in preformed pools, and instead the synthesis and expression of this selectin is initiated by cytokines, such as TNF-α and IL-1β, and LPS. When neutrophils are activated, such as in sepsis or endotoxemia, they shed their L-selectin and increase their expression/activation of the β2 integrins (CD11/CD18). These integrins engage with ligands such as ICAM-1 (also known as cluster of differentiation (CD54)), and VCAM-1 (also known as vascular cell adhesion protein 1 or of differentiation 106 (CD106)) on the activated endothelium.⁶⁷ Septic patients have increased soluble adhesion molecules, such as sE-selectin, sP-selectin, sVCAM-1, and sICAM-1.⁶⁸ Although studies have been performed to address whether any of these soluble factors could be a measure of disease severity, or a predictor of MODS and/or survival, it would appear that due to the transient nature of sE-selectin (which peaks at 6–8 h, returning to baseline levels within 24 h), this adhesion molecule might be a good predictor of patients who are at risk for becoming septic and developing MODS,⁶⁵,⁶⁹,⁷⁰ and sICAM-1 (which remains elevated for up to 24 h on activated endothelium (93,134)) may be a better predictor of severity of sepsis and mortality.⁷¹,⁷²

Following successful recruitment to the endothelial luminal cell surface, leukocytes then transmigrate either in a paracellular fashion, through reorganization and utilization of interendothelial junction molecules or by a transcellular route, involving the formation of vesiculo-vacuolar organelles consisting of ICAM-1-containing caveolae.⁷³

In sepsis, activated neutrophils transmigrate and infiltrate into the tissues, such as the lungs, releasing MPO, and ROS. Recently, it has been demonstrated that the integrin α3β1 (VLA-3; CD49c/CD29) is dramatically upregulated on neutrophils isolated from both human septic patients and in mouse models of sepsis,⁷⁴ and blocking of α3β1 may represent a new therapeutic approach in sepsis treatment.

Nuclear factor-kB (NF-kB), which is believed to be the main primary transcription factor involved in the regulation of both cytokine and adhesion molecule expression,³³ is activated by LPS (and the production of cytokines, such as TNF-α and IL-1). NF-kB itself also transactivated various proinflammatory genes, which leads to increased cytokine production and adhesion molecule expression. LPS increases NF-kB mobilization both at the whole organ⁷⁵–⁷⁷ and cellular⁷⁸,⁷⁹ level,³³ which has been demonstrated in vivo, with activation of NF-kB being directly associated with increased expression of cytokines,⁷⁶,⁸⁰ adhesion molecules,³³ chemokines,⁷⁷,⁷⁸ and iNOS.⁸⁰

Platelets in sepsis

Platelets play a significant role in sepsis, and in critically ill patients, a 30% drop in platelet count independently predicts death. Briefly, platelet–endothelial interactions, platelet aggregation, and microthrombi formation in capillaries can all plug capillaries and therefore impair blood flow. Tissue factor (TF) initiates DIC, leading to thrombosis. LPS also increases platelet cell surface expression of adhesion molecules, for example, PSGL-1 and P-selectin, which leads to platelet adhesion. Thus, in sepsis, platelet activation, aggregation, and platelet–endothelial adhesion leads to microthrombi and capillary no reflow. Furthermore, activation of leukocyte function-associated antigen 1 (LFA-1) on platelets also targets bacteria, by triggering the release of neutrophil extracellular traps (NETs), primarily composed of DNA and extracellular fibers, which promote thrombus formation and trap free bacteria (Fig. 1.1). See Chapters 2 and 3 for further details.

Thrombosis and coagulation in sepsis

Microvascular plugging is increased in septic patients, in part due to both red and white blood cell deformability. Red blood cells (RBCs, which have a diameter of 8 μm and a thickness of 2 μm in humans) not only transport oxygen, but also play an important role in regulating microcirculatory oxygenation, by their ability to sense hypoxia and release vasodilators, for example, NO and ATP.⁸¹ These mechanisms help to control blood flow and supply the tissues with the oxygen. Direct diffusion of oxygen from arterioles to other vessels with lower oxygen content, thus bypassing the capillaries, also contributes to this process of oxygen supply.⁸²

Even though leukocytes (e.g., neutrophils, which have a diameter of 6–8 μm in humans) are much less deformable than the RBCs, they still play an important role in coagulation and thrombosis, through their augmented binding to activated microvascular endothelium and through the formation of leukocytic aggregates, which ultimately decreases oxygen delivery.⁸³,⁸⁴ Large neutrophil numbers are found sequestered and aggregated in organs undergoing failure, as evidenced in autopsy specimens from patients with MODS, which revealed excessive amounts of neutrophils in both renal blood vessels⁸⁵–⁸⁷ and in the lung.⁸⁸ Thus, leukocyte adhesion not only increases blood flow resistance and impairs RBC flow in microvessels, but also increases microvascular permeability, further contributing to tissue edema and decreased circulating volume.⁸⁹

As mentioned earlier, the TF pathway is the primary coagulatory pathway that drives coagulation and thrombosis in sepsis. In particular, there is increased endothelial cell TF release, decreased thrombomodulin expression, and, therefore, decreased protein C activation (low levels of protein C have been related to poor outcomes in septic patients),⁹⁰ and abundant intravascular fibrin formation; all of which culminates in widespread ischemic organ damage, organ necrosis, and MODS. Of interest, patients suffering from fulminant meningococcal sepsis have presented with small-vessel thrombosis in their limbs, which has been associated with the loss of thrombomodulin, an endothelium-based anticoagulant.³ However, as discussed later, despite many nonclinical studies finding the beneficial effects of targeting various molecules in the coagulation/thrombosis pathway, for example, TFPI, ATIII, heparin, and activated protein C (rAPC), only rAPC showed benefit in a phase 3 trial.

This prothrombotic phenotype assumed by inflamed tissue in sepsis and endotoxemia also reflects an interdependency of coagulation and inflammation (Fig. 1.1). For example, we and other groups, have shown the importance of one particular signaling pathway, the CD40/CD40L, as a contributor to the inflammation-enhanced thrombus formation in the microvasculature, in both endotoxemic models and other inflammatory models.⁹¹–⁹³ CD40 is expressed on vascular endothelial cells, and its expression is increased by acute and chronic inflammatory stimuli, such as LPS⁹³,⁹⁴ and DSS-induced colitis.⁹³,⁹⁵ CD40L is a membrane glycoprotein (GP) belonging to the TNF family, and is expressed by activated T cells and platelets. CD40L is shed from the cell surface (mainly from platelets), and its concentration in plasma has been used as a prognostic marker for inflammation and thrombotic risk in human disease.⁹³,⁹⁶,⁹⁷ We found that the protection against thrombosis afforded by genetic deficiency of either CD40 or its ligand in an endotoxemic model, supports the development of therapeutic strategies that target either or both components of CD40/CD40L signaling to reduce the thrombotic risk associated with acute and chronic inflammation.⁹³

Models

A number of different models have been employed to study the pathophysiology of sepsis. The most commonly used are the induction of endotoxemia by administration of LPS and the induction of peritonitis by cecal ligation and puncture (CLP) model. There are pros and cons of each, for example, the LPS model had been criticized that it lacks some of the clinical manifestations, and the doses used often exceed those that would be found in the patient (in one study the LPS dose required to produce similar cytokine responses as those seen clinically, was 250 times higher in mice than in humans).⁹⁸ On the other side, the CLP model, while clinically more relevant,⁹⁹,¹⁰⁰ there are problems associated with the amount of fecal contamination in the abdominal cavity, and the assessment of endpoint post-CLP induction has led to variations in results. Despite the pros and cons of both these models, many concepts in sepsis have been discovered.

It is also important to mention that LPS (or endotoxin) tolerance has been discovered to occur when animals or cells are pretreated with LPS (even a high-dose), as it induces a refractory state in cells, and renders them more resistant to a subsequent LPS challenge.¹⁰¹ However, low dose (in the picogram/milliliter range) LPS/endotoxin primes the cells/animals into a proinflammatory state to subsequent endotoxin challenge – a phenomenon known as the Shwartzman-like reaction.¹⁰²

With respect to measuring microcirculatory blood flow (either directly or indirectly) in sepsis, a variety of modalities are now available, including (intravital) microscopy/videomicroscopy, fluorescence videomicroscopy, orthogonal polarization spectral imaging, sidestream dark-field imaging, and flowmetry. Each modality has its own pros and cons for use, and while it is beyond the scope of this chapter, several excellent reviews are available.¹⁰³,⁴⁷

In summary, scientists and clinicians have gained a good insight into sepsis and the pathophysiology of this disease using these different animal models and approaches. With time these approaches will evolve, which will hopefully lead to better therapeutic targets and treatments for patients with sepsis.

Genetics and sepsis

Increased susceptibility to infection, including sepsis, and poor outcomes in patients, have been attributed to a number of different genetic polymorphisms. In sepsis, the risk of mortality is increased in patients with polymorphisms in the genes for cytokines and chemokines, such as IL-10, IL-18, IL-6, and TNF-α.¹⁰⁴–¹⁰⁹ Polymorphisms have also been associated with disease outcome in other forms of sepsis, such as fulminant meningococcal sepsis in which polymorphisms in Fc receptors for IgG have been found.¹¹⁰

Treatments for sepsis

Despite the ever-growing clinical and nonclinical research into sepsis, this clinical issue still remains a serious problem. Most treatment for sepsis and related organ dysfunction is palliative, with a wide range of therapeutic strategies having failed, such as antagonizing TNF-α activity, high-dose corticosteroids, or the recent trial that focused on the administration of APC as a therapeutic intervention (which failed due to the lack of improvement in the outcome of patients with sepsis and MODS).¹¹¹–¹¹⁴ Other options for treatment include controlling the source of infection by performing antimicrobial (e.g., antibiotics) therapy, gaining hemodynamic support by administering vasopressor drugs and fluid resuscitation, and providing adequate nutrition.¹¹⁵ Other potential targets for treatments include targeting microvascular dysfunction, by the administration of drugs that improve function, such as antioxidants and TFPIs. For a more in-depth look into clinical treatments see Chapter 17.

Conclusions

In summary, sepsis is a complex life-threatening clinical problem, triggered by an infection (most commonly bacteria, such as LPS, but also parasites, viruses, and fungi). It results in microvascular dysfunction via dysregulation of vasomotor control, endothelial injury, coagulation activation, and disordered leukocyte trafficking. While early treatment of the disease (often with antibiotics and large amounts of intravenous fluids) improves survival, it is most common and most dangerous in older adults or those with weakened immune systems. Taken together, it is clear that all elements of the microcirculation are involved, including arterioles, capillaries and venules, and further investigations are very much needed to understand the pathophysiology of this infection, which leads to organ failure and death.

Acknowledgment

I should like to thank Dr Helen K. Smith (LSUHSC-S) for her artwork in Fig. 1.1.

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