Principles of Vascular Toxicology

Mitchell Troutman, D.V.M.

The University of North Carolina at Chapel Hill

Basic Vascular Structure:Endothelium

Smooth Muscle

Connective Tissue

Basic Vascular Function

• Arteries, veins, and capillaries

– Conduct blood to tissues– Transport

• oxygen• nutrients• wastes• drugs/chemicals (endogenous and

exogenous)

• Lymphatics– Return interstitial tissue fluid to blood

Endothelial Cell Properties & Functions

• Maintenance of permeability barrier• Anticoagulant/antithrombotic molecules

– prostacylcin, thrombomodulin, plasminogen activator (PA), heparin-like molecules

• Prothrombotic molecules– von Willebrand factor, tissue factor, PA inhibitor

• Extracellular matrix production– collagen, proteoglycans

• Modulation of blood flow & vascular reactivity– vasoconstrictors (endothelin, ACE {Angiotensin

converting enzyme})– vasodilators (nitric oxide, prostacyclin)

• Regulation of inflammation & immunity– adhesion molecules, interleukins, histocompatibility

antigens

• Regulation of cell growth– growth stimulators (PDGF, CSF, FGF)– growth inhibitors (heparin, TGF-)

• Oxidation of low-density lipoprotein (LDL)

Endothelial Cell Properties & Functions

Vascular Smooth Muscle Cells

• Vasoconstriction/vasodilation– in response to normal or pharmacologic

stimuli

• Synthesis of connective tissue– collagen, elastin, proteoglycans

• Release of growth factors & cytokines

• Migration & proliferation

Manifestations of Vascular Dysfunction

• Structural– arteriosclerosis/atherosclerosis– vasculitis– prothrombotic events

• Functional (physiologic)– hypertensive arteriosclerosis– prothrombotic events

Animal Models

Rat very resistant to development of atherosclerosis;

classic model for studying hypertension

Rabbit sensitive to microvascular constriction induced by release of epinephrine and norepinephrine

Dog very similar to human electrophysiology; resistant to development of atherosclerosis

Swine naturally develops high incidence of atherosclerosis

Primates rhesus is sensitive to development of extensive atherosclerosis on high-cholesterol diets

Atherosclerosis

Normal Coronary Artery Coronary Artery with plaque formation and narrowing of vascular lumen

• Response to injury hypothesis– injury to arterial endothelium (inc. hypertension)– increased permeability to plasma constituents– adherence of blood monocytes (+ migration) and

platelets– lipid accumulation in macrophages foam cells– migration and proliferation of smooth muscle cells

deposition of collagen and proteoglycans– repeated injury atheromatous plaque forms

Pathogenesis of Atherosclerosis

                                                                       

Atheromatous Plaque

Atherosclerosis: Importance in Toxicology

• Toxicants can accelerate or exacerbate– cadmium, selenium, chronic copper toxicity– carbon monoxide– cholesterol and oxygenated derivatives– homocysteine

• Major therapeutic area of interest

Hypertension (high Blood Pressure)

• Influenced by many factors– cardiac output, vascular compliance, renal

disease

• Blood vessels become thickened and less distensible heart works harder to circulate blood

• Chronic damage and cardiovascular failure can occur

Hypertensive Arteriosclerosis

• Pathogenesis:– inadequate renal sodium excretion

increased neurohormonal release

– increased plasma and ECF volume

– increased cardiac

output

HYPERTENSION that leads to chronic vascular endothelial

damage

increased natriuretic hormone

increased total peripheral resistance

Hypertensive Arteriosclerosis

Canine glomerular hyaline arteriosclerosis, PAS stain, 200X

Hypertensive Arteriosclerosis

Normal Coronary Artery Coronary Artery branch with hyperplastic arteriosclerosis and near occlusion of vascular lumen

• Toxicants can accelerate or exacerbate– cadmium, arsenic, mercury– allylamine, chlorophenoxy herbicides– organophosphates

• Can accelerate atherosclerosis and potentiate cerebrovascular hemorrhage

Hypertensive Arteriosclerosis: Importance in Toxicology

What is drug-induced vasculitis?

• Vasculitis: inflammation of blood vessels

• Vasculitis can occur either:1. Spontaneously – Canine polyarteritis

nodosa (i.e., canine pain syndrome)

2. After exposure to exogenous agents

- Infection agents

- Pharmaceuticals (i.e., drug-induced)

Drug-induced vasculitis: In Humans• Most drug-induced vasculitides are non-necrotizing

hypersensitivity type (e.g., binding of drug/hapten + protein/carrier antigen), involving small and medium arteries and veins and spare large arteries. Localized mainly to the skin (maculopapular rashes followed by palpable purpura), often associated with systemic symptoms (e.g., arthralgia, malaise, fever); end-stage organ damage (e.g., lungs, kidneys) possible from immune-complex deposition

Examples:• Many drugs ( e.g., antibiotics, propylthiouracil, hydralazine)• Biologics (e.g., hematopoietic growth factors, interferons,

mAbs)

ANIMALS ARE NOT GOOD PREDICTORS OF IMMUNE-MEDIATED VASCULITIS IN HUMANS

Drug-induced vasculitis: In animals

• Most common is: Due to drug- induced hemodynamic alterations (vasoactive arteriopathy). Usually involves medium to large sized vessels, resulting from functional damage associated with excessive hemodynamic activity

• Less common are:– Due to exacerbation of spontaneous disease– Due to primary cytotoxicity (toxic vasculitis, like

allylamine and acrolein)– Due to immune-mediated mechanism (hypersensitivity

vasculitis), affecting thin-walled vessels, sparing muscular arteries

End-stage organ damage is rare

Drug-induced hemodynamic alterations (vasoactive arteriopathy)

Pharmacological agents inducing vasoactive arteriopathy are divided into:a. Vasodilators and positive inotropic agents – Pathology: Rat – Splancnic, renal and ovarian large muscular arteries Dog - coronary vessels Examples are as follow: Dopaminergics agonist – Fenoldopam, dopamine Dopamine ß-hydroxylase inhibitors – SKF 102698 Phosphodiesterase (PDE) inhibitors - theophylline Serotoninergic compounds – SKF 103829 Endothelin A receptor antagonists – bosentan

Possible mechanisms: Prolonged vasodilatation associated with increased regional blood flow and vessel wall stress

b. Vasoconstrictors – Endothelin-1, noradrenaline and digoxin (coronary arteritis in dog) Monocrotaline (pulmonary arteritis in rats)

Theophylline (a nonspecific PDE inhibitor)

induce splanchnic arteriopathy in rats

Nyska A, Herbert RA, Chan PC, Haseman JH, Hailey JR (1998) Theophylline-induced mesenteric

periarteritis in F344/N rats.

Arch. Tox. 72:731-737

Theophylline (1,3-dimethyxanthine)

• An alkaloid found in cocoa and tea• Structurally related to caffeine and theobromine• Used as bronchodilator such as in asthma and

myocardial stimulation• In the NTP studies - The compound was

administered by gavage to B6C3F1 mice and F344 rats, but vascular lesions were seen only in rats (mainly males) in the 16-D, 14-W and 2-Y, at doses of 75 mg/kg

• Vascular lesions affected the medium and large splanchnic arteries

Hemorrhage and necrosis within the media of a mesenteric artery of male rat given 400 mg theophylline/kg BW for 16 D

Hemorrhage (H) and necrosis (N) within the media of a mesenteric artery of male rat given 400 mg theophylline/kg BW for 16 DNote the damage is located at arterial bifurcation (mesenteric artery and 1st branch)

N

N

H

H

Periarteritis in pancreatic arteries of male F344 rat given 75 mg/kg of theophylline

for two years.

Thickened wall

Thickened media (hypertrophic smooth muscle cell and fibrosis)

Thickened adventitia (fibrosis)

vasoactive arteriopathy Mechanistic study

Phosphodiesterase III inhibitor-induced mesenteric arteriopathy in

rats

Ref: Joseph EC, Rees JA, Dayan AD. (1996). Mesenteric arteriopathy in the rat induced by

phosphodiesterase III inhibitors: an investigation of morphological, ultrastructural, and hemodynamic

changes.Toxicol Pathol. 1996 Jul-Aug;24(4):436-450.

SK&F 95654 (PDE III inhibitor)-induced arteriopathy model in rat – pathogenetic

investigation• Used as potent inotropic/vasodilator • Pharmacologically: PDE III inhibition increases

cAMP in the arterial smooth muscle

PATHOLOGY:• Dogs: Epicardial arteriopathy• Rats: Focal or segmental medial necrosis or

hemorrhage in splanchnic arteries (100-800 micron in diameter)

• Objective: Follow the development of time-course (up to 24 hours) changes in peripheral systolic blood pressure and splanchnic arterial lesions

Drug-induced decrease in systolic blood pressure over 24h following subcutaneous administration of Vehicle control (, DMSO) and SK&F 95654 at 0.174mmol/kg () and 0.697mmol/kg ().

high dose

control

Time-course development of histopathological and SEM findings• 1st changes after 6 h – endothelial raising and

pronounced interendothelial projections• 12 h postdosing – medial hemorrhage and

medial compression, degeneration and necrosis • 16 h postdosing – endothelial necrosis,

adhesion of leukocytes and activated platelets• 24 h postdosing – medial necrosis and infiltration

of inflammatory cells with RBCs

Mesenteric arcade from a rat showing severe grade lesion. Multiple large foci of Hemorrhage are present on all first-branch arteries. Note absence of hemorrhage in superior mesenteric artery (sm) but presence of lesion at junction with first-branch

first branches of mesenteric artery

Large intestine

Smallintestine

Macroscopic photograph of first-branch mesenteric artery from a rat showing Severe Grade hemorrhagic lesion

first branch of mesenteric artery

SEM, normal endothelial surface of first-branch mesenteric artery from an untreated rat showing confluent layer of interdigitating endothelium (E) with clearly defined plasmalemma ridges.

SEM, focal interendothelial gaps formation - endothelial surface of first-branch mesenteric artery from rat 6 hr following sc does of 0.697 mmol/kg SK&F 95654 showing raised endothelium (E) and multiple interendothelial projections

Pathogenesis and comparative aspects

The study suggests that:

• Interendothelial gaps are consequence of passive stretching of the endothelium as a result of vasodilatation and associated increased intramural tangential stress

• The damage occurs when the critical intramural tension has been exceeded

• There was a close correlation between the magnitude and duration of hypotension and severity of arterial lesions

Joseph EC (2000). Arterial lesions induced by phosphodiesterase III (PDE III) inhibitors and DA(1) agonists. Toxicol Lett. 15;112-113:537-46

What does drug-induced vasculitis in animal studies mean to patients (extracted from ”Drug-induced vasculitis: FDA’s perspectives”, presented by Thomas

Papoian, Senior pharmacologist FDA, at Toxicology Forum, Aspen, July, 2004 (http://www.toxforum.com)

• Possibly nothing, if vascular effects in animals do not occur in patients under therapeutic conditions or exposure

- Theophylline showed vasculitis in rats, but has been prescribed in human for many years without any apparent vascular toxicity

- (assuming that post-marketing surveillance mechanisms capable of detecting a drug-related signal in people with cardiovascular disease)

What does it mean to patients (extracted from ”Drug-induced vasculitis: FDA’s perspectives”, presented by Thomas Papoian, Senior

pharmacologist FDA, at Toxicology Forum, Aspen, July, 2004 (http://www.toxforum.com )– Cont.

• But if these specific drug-induced vascular effects can or do occur in patients, then there may be a significant cause for concern because:

- Vascular inflammation predisposes to:= progression of atherosclerosis (considered an inflammatory

disease)= Rupture of vulnerable plaques= Increased incidence of cardiovascular events: heart attack,

stroke, or death- No good way to monitor for vascular inflammation in patients

(i.e., no validated specific biomarkers) THE QUESTION: Can certain drugs found to produce

vasculitis in animals contribute to the arteriosclerotic process in human?

Approved drugs that cause vascular (vasoactive) injury

• Minoxidil• Adenosine• Hydralazine• Milirone• Cilomilast (PDE IV)• Fenoldopam• Bosentan• Theophylline• Caffeine• Nocorandil

Particulate-induced vasculitis and cardiomyopathy

Particle matter air pollution size distributionParticles are classified according to their median aerodynamic diameter:Thoracic particles- PM10-deposit in the upper tracheobronchial treeCoarse fraction – PM 10 to 2.5 (10 to 2.5 μM - nanometer). Predominantly natural sources – as soil and grinding Fine particles - PM 2.5 (<2.5 μM). Originate from combustion sources, include primary and secondary particlesUltrafine particles – PM 0.1 (<0.1 μM). Originate from combustion sources, deposits in the alveoli, able to pass directly to the circulatory system from the alveoli

The triggering effect of air pollution in coronary

atherosclerosis and acute myocardial infarction • Atherosclerosis is inflammatory – degenerative disease of the arteries• Initiating atherosclerotic factors are for example, disturbed coronary

blood flow and low shear stress• The air pollution may trigger the atherosclerosis process, and cause

the rupture of quiescent focal atherosclerotic lesion (vulnerable plaque disruption) by inducing or exacerbating one or more of the following effects:

1. Pro-thrombosis effect (e.g., increased fibrinogen, increased viscosity of the plasma, platelet activation)

2. Vasoconstrictive effect due to release of the vasoconstrictors (endothelins)

3. Pro-inflammatory effect (e.g., local or systemic inflammation via action of cytokines, chemokines and Reactive Oxygen Species)

Extracted from: Review : Potential Role of Ultrafine Particles in Associations between Airborne Particle Mass and Cardiovascular Health. Ralph J. Delfino, Constantinos Sioutas, and Shaista Malik.EHP, August 2005

Toxicologic Pathology 2002; 30: 427-434

Introduction

Epidemiological studies demonstrated an association between exposure to high levels of ambient particulate matter (PM) and increased morbidity and mortality of cardiopulmonary disease in human

Findings in NTP studies with particulates

inhalation exposure for 2 years of B6C3F1 mice

• Highly significant association between exposure to indium phosphide and cobalt sulfate heptahydrate (particulate size 1.1-1.8

micron) and incidences of coronary and renal arteritis

• Marginal significant association between exposure vanadium pentoxide and gallium arsenide (particulate size 1.1-1.8 micron) and incidences of coronary and renal arteritis

Aspects of indium phosphide- induced arteritis in mice - mononuclear cell arteritis, fibrinoid necrosis, smooth muscle proliferation

Suggested mechanisms

• Hypoxia (severe lung space occupying pathology)

• Autonomic nervous system activation leading to changes in heart rate (conductive arrythmia and depression in heart rate) or modulation of the coronary vascular tone inducing vasodilation as in the case of PDE III inhibitors

• Changes in plasma viscosity (increased plasma fibrinogen)

• Increased cytokine expression in the lungs (IL-6)

Chemically-induced prothrombotic events

2-Butoxyethanol

• Ethylene glycol monobutyl ether

• A major environmental chemical intermediate used in the manufacturing of a wide range of domestic and industrial products.

• Chemical formula = C6H14O2

H3C

H2

CCH2

H2

CO

H2

CCH2

OH

Objective

To characterize the acute and protracted histological and x-ray

alterations in bones from rats treated with 2-butoxyethanol.

Study design

Group TreatmentDay of

Sacrifice

1 Control Day 4

2 250mg/kg BE Day 4

3 300mg/kg BE Day 4

4 Control Day 30

5 250mg/kg BE Day 30

6 300mg/kg BE Day 30

Hematological Findings

• Marked hemolysis • Female more sensitive than males. Peak reduced

RBC in females after 3 doses, and in males, after 4 doses

• Significant alterations in the morphology of RBCs such as stomatocytosis, spherocytosis, schistocytosis, and massive fragmentation.

• Presence of numerous nucleated RBCs, such as rubricytes and metarubricytes

• Increased numbers of platelets

Blood Smears from Female Rats Showing the Time-Related Morphological Effects of BE

Main Characteristics of the Hemolytic Anemia: Reduced Mature RBC, Marked Variation in RBC Size (Anisocytosis); Regeneration (Polychromatophilia); Platelet Aggregation

Untreated Control

Spherocytes

Metarubricytes

Ghost Cells

Stomatocytes

Polychromatophilia

Platelet Aggregation

3-Doses 4-Doses

2-Doses

Stomatocyte

Schistocytes

Effect of 2-butoxyethanol treatment for 4 days at 250 mg/kg/day

Tip of tail infarction

Thrombosis

• Heart• Lung• Femur• Tooth• Tail• Nasal mucosa• Liver• Eye

Cortical bone thrombosis, osteocyte necrosis, and bone marrow necrosis, following 2-butoxyethanol treatment for 4

days at 300 mg/kg/day.

FemurTreated Control

Note the radiolucency in the diaphysis of the treated animal as shown by the red arrows.

Tail Vertebra - H&E

Note the infarction of the growth plate and granulomatous inflammation in the marrow space of this treated animal.

The Pathogenesis of BE-Induced Disseminated Thrombosis is Uncertain,

Several Mechanisms Have Been Proposed:

• Release of procoagulant factors from destroyed erythrocytes or other sources

• Disturbed blood flow secondary to changes in erythrocytic rheological properties (i.e. self-aggregability, deformability and adherence to blood vessel wall endothelium)

• Direct endothelial damage

The End!