Transcript Principles of Vascular Toxicology Mitchell Troutman, D.V.M. The University of North Carolina at Chapel Hill.

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
Endothelial Cell Properties &
Functions
• 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)
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
Pathogenesis of Atherosclerosis
• 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
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 natriuretic hormone
– increased cardiac
increased total
output
peripheral resistance
HYPERTENSION that leads to
chronic vascular endothelial damage
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
Hypertensive Arteriosclerosis:
Importance in Toxicology
• Toxicants can accelerate or exacerbate
– cadmium, arsenic, mercury
– allylamine, chlorophenoxy herbicides
– organophosphates
• Can accelerate atherosclerosis and
potentiate cerebrovascular hemorrhage
What is drug-induced vasculitis?
• Vasculitis: inflammation of blood
vessels
• Vasculitis can occur either:
Spontaneously – Canine polyarteritis
nodosa (i.e., canine pain syndrome)
2.
After exposure to exogenous agents
- Infection agents
- Pharmaceuticals (i.e., drug-induced)
1.
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
N
H
N
H
Hemorrhage (H) and necrosis (N) within the media of a mesenteric
artery of male rat given 400 mg theophylline/kg BW for 16 D
Note the damage is located at arterial bifurcation (mesenteric artery and 1st branch)
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 inhibitorinduced 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
control
high dose
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 ().
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
Large intestine
first branches of mesenteric artery
Small
intestine
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 branch of mesenteric artery
Macroscopic photograph of first-branch mesenteric artery
from a rat showing Severe Grade hemorrhagic lesion
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
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Minoxidil
Adenosine
Hydralazine
Milirone
Cilomilast (PDE IV)
Fenoldopam
Bosentan
Theophylline
Caffeine
Nocorandil
Particulate-induced vasculitis and cardiomyopathy
Particle matter air pollution size distribution
Particles are classified according to their median aerodynamic diameter:
Thoracic particles- PM10-deposit in the upper tracheobronchial tree
Coarse 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 particles
Ultrafine 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
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1.
2.
3.
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:
Pro-thrombosis effect (e.g., increased fibrinogen, increased viscosity
of the plasma, platelet activation)
Vasoconstrictive effect due to release of the vasoconstrictors
(endothelins)
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
H2
C
H3C
H2
C
C
H2
H2
C
O
OH
C
H2
Objective
To characterize the acute and
protracted histological and x-ray
alterations in bones from rats
treated with 2-butoxyethanol.
Study design
Group
Treatment
Day 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
Spherocytes
Schistocytes
Metarubricytes
2-Doses
Untreated Control
Ghost Cells
Stomatocytes
Platelet Aggregation
Polychromatophilia
3-Doses
Stomatocyte
4-Doses
Main Characteristics of the Hemolytic Anemia: Reduced Mature RBC,
Marked Variation in RBC Size (Anisocytosis); Regeneration (Polychromatophilia);
Platelet Aggregation
Effect of 2-butoxyethanol treatment
for 4 days at 250 mg/kg/day
Tip of tail infarction
Thrombosis
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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.
Femur
Treated
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!