Drug-Induced Vascular Injury---A Search for Biomarkers

**Discussion Document for the NonClinical Studies Subcommittee**

of the Advisory Committee on Pharmaceutical Science

**By**

**The Expert Working Group on Drug-Induced Vascular Injury September 9,
2002**

**Expert Working Group Members:**

> William Kerns-Pharma Consulting Inc. (Co-Chair)
>
> Lester Schwartz-GlaxoSmithKline (Co-Chair)
>
> Kerry Blanchard-Boehringer-Ingleheim
>
> Scott Burchiel-University of New Mexico
>
> Robert Johnson-Schering-Plough
>
> Fred Miller-NIEHS
>
> Prakash Nagarkatti-Virginia Commonwealth University
>
> Don Robertson-Pfizer
>
> Paul Snyder-Purdue University

**Active Contributors:**

> Eric Fung-Ciphergen
>
> Mike Lawton-Pfizer
>
> Calvert Louden- Astra-Zeneca
>
> Heath Thomas-GlaxoSmithKline
>
> Anthony Ward-BD Biosciences

**FDA Liaisons:**

James MacGregor-NCTR

Frank Sistare-CDER

David Essayan-CBER

Thomas Papoian-CDER

**Drug-Induced Vascular Injury --- A Search for Biomarkers**

**Introduction.** Arterial and venular injury is a relatively common
hazard identified during nonclinical toxicity testing within certain
pharmacological classes of drug candidates. Drugs that induce vascular
lesions in animals present a safety assessment dilemma to toxicologists,
physicians and regulators wishing to assess the safety of new medicines
for humans. This dilemma is confounded by the gaps in our knowledge
regarding pathogenesis of drug-induced vascular injury in animals and
importantly, the absence of validated nonclinical or clinical methods
for monitoring vascular integrity noninvasively.

Contrary to past thinking, nonclinical experience with new and novel
pharmaceuticals suggests that vascular injury is not always associated
with systemic changes in blood pressure and heart rate, rendering these
parameters of little value, clinically. Variation in species
responsiveness to vasoactive and non-vasoactive agents and marked
differences in reactivity of selected vascular beds, taken together with
contributions from numerous reactive cell types (e.g., endothelium,
vascular smooth muscle, and inflammatory/immune cells) all add
complexity to defining mechanism(s) and defining robust biomarkers.

Experimental evidence suggests that drug-induced vascular injury in
animals can occur via one or more of the following overlapping
mechanisms:

-   Biomechanical injury following changes in shear and/or hoop stress.

-   Toxicity following direct pharmacological and/or chemical
    perturbation.

-   Injury via immune-mediated mechanisms.

Historically, toxicology studies in normal animals have not predicted
the occurrence of the drug-induced hypersensitivity vasculidities, which
are by far the most common drug-induced adverse vascular responses
observed in humans. In general, drug-induced hypersensitivity angitis of
humans is not recognized in toxicology studies in healthy animals.
Conversely, drug-induced vascular lesions recognized in animals are not
known to occur in humans, however this could be related to our lack of
knowledge and methods for monitoring in humans. Until these methods
become available, it will be difficult to make claims of species
specificity. Confusion in histological terminology and lack of
consistency in characterizing the key components of vascular damage have
resulted in communication gaps between pathologists, toxicologists,
regulators and clinicians, all who share responsibility for ultimately
making a judgment on human risk. Poor understanding of pathogenic
mechanisms, limitations of animal models and lack of valid biomarkers
for nonclinical and clinical monitoring has elevated this issue to a
shared level of concern between FDA and the pharmaceutical industry.

**EWG Mandate.** To evaluate the current state of knowledge of
drug-induced vascular injury in animals and humans and to identify
opportunities for the discovery and validation of predictive biomarkers
of acute vascular injury that could be used in nonclinical drug safety
programs and eventually in Phase I/II clinical programs.

**White Paper Objectives.** The purposes of this white paper are:

1.  review the current status of the field

2.  present current understanding on mechanisms of drug-induced vascular
    injury

3.  identify opportunities for practical biomarkers and high potential
    research areas

4.  annotate emerging technologies holding promise in the search for new
    markers

5.  identify opportunities that may be met through collaborative work
    between public and private partners

6.  confirm that this is an issue that needs resolution

# MECHANISMS OF DRUG-INDUCED VASCULAR INJURY

## Introduction

Limited understanding of the mechanisms by which drugs cause vascular
injury has been a significant barrier to progress in the development of
reliable biomarkers of vascular injury. This facet is further confounded
by studies indicating multiple and distinct mechanisms operating in
these syndromes. This is not entirely surprising because a wide variety
of drugs, with varied pharmacologic effects that have been implicated,
could potentially act on different cells but trigger a common
down-stream event leading to vascular injury. A review of the literature
indicates that at least three major mechanistic pathways may contribute
to drug-induced endothelial and smooth muscle compromise, resulting in
vascular injury in animals. These mechanisms can be broadly classified
into the following categories:

-   Biomechanical injury following changes in shear and/or hoop stress

-   Toxicity following direct pharmacological and/or chemical
    perturbation

-   Injury via immune-mediated mechanisms

Such mechanisms are not mutually exclusive and in fact there could be
significant overlaps between these routes of injury.

### Biomechanical Injury

The relevance and uncertain extrapolation of drug-induced arterial
toxicity in rats, dogs and other species to humans continues to be an
obstacle to drug-discovery and development, as well as a concern to
regulatory scientists. The issue of arterial toxicity particularly in
the dog is further complicated by the occurrence of spontaneous coronary
arterial lesions that are reported to mimic drug-induced vasculopathy
(22) and the inability of modern science and technology to identify
clear and unequivocal markers, suitable for assessment and monitoring of
potential drug-related vascular injury in animals and humans.
Historically, in the dog, vascular toxicity has been associated with
profound cardiovascular hemodynamic changes in mean arterial pressure
(MAP) and heart rate (HR), which have been used as surrogate markers of
potential vascular toxicity in man at therapeutic doses (24, 66-68).
However, recent experiences with a novel class of vasoactive agents,
endothelin receptor antagonists (ETRAs) and others, suggest that
profound hemodynamic changes (MAP and HR) are not a pre-requisite for
development of coronary arterial lesions in the dog (3, 58). For this
class of molecules, hemodynamic monitoring as a method to assess the
potential hazard to humans is not possible and extrapolation of the
potential risk to the human population can only be made on the basis of
canine responses and the therapeutic index in the dog (3, 24, 58,
66-68). This species-sensitive response in the dog has yet to be
supported by scientific data that describes a unique mechanism of
vascular toxicity that is not applicable to man at therapeutic
concentrations.

A number of structurally diverse pharmacological agents can induce
arterial lesions in rats and dogs, and the mesenteric and coronary
arteries, respectively, are primarily affected. The reasons for this
tissue-specific selectivity are not clear but may be related to
increased sensitivity in these respective vascular beds because
spontaneous arterial lesions develop at these sites in both species (22,
38, 45, 50). The beagle dog used routinely in toxicology studies is
considered to be an extremely sensitive species to drug-induced coronary
arterial lesions. For example, adenosine agonists and an ETRA will
induce coronary arterial lesions in dogs and monkeys (2-4)). However, in
the dog lesions occur after a very short duration of treatment at lower
systemic exposure compared to the monkey. The mechanisms of this
selective species response are ill-defined but may be related to
localized changes in blood (42) flow as demonstrated with adenosine (3,
7, 9, 10), an endogenous substance released from the heart, adenosine
agonists, (61, 69, 92) and a number of structurally diverse vasoactive
pharmacological agents (24, 58). In the rat, there is indirect evidence
linking increases in localized mesenteric blood flow and development of
drug-induced mesenteric arterial lesions (48, 49). Therefore,
exploration of the contributions of localized changes in blood flow (BF)
and subsequently changes in shear and hoop stress, to drug-induced
arterial injury is worthy of investigation in both animals and humans.

#### Flow-mediated mesenteric arterial lesions in the rat {#flow-mediated-mesenteric-arterial-lesions-in-the-rat .unnumbered}

Within the mesenteric arteries, physiological activation of specific
dopaminergic receptor subtypes (DA1) by fenoldopam leads to marked
vasodilation and mesenteric arterial lesions (49). In order to test the
vasodilation hypothesis, fenoldopam was administered with DA1 receptor
antagonist and this treatment regimen prevented induction of hemorrhagic
lesions in rats (49). Other studies have also reported that
co-administration of the vasoconstrictor methoxamine attenuated the
fenoldapam induced splanchnic arterial lesions. The vasodilation
hypothesis was also tested using SK&F 95654, a potent Phosphodiesterase
Inhibitor (PDEIII) which causes marked reduction in mean arterial
pressure (\~40 %) and induction of mesenteric arterial lesions.
Co-administration of arginine vasopressin, a vasoconstrictor, reversed
the PDE hypotensive effect in a dose-dependent manner and prevented
induction of mesenteric arterial lesions (46).

When administered at doses that induce mesenteric arterial lesions, SK&F
95654 a potent PDEIII inhibitor and Minoxidil a potent K+ channel opener
produced long-lasting increases in mesenteric blood flow for 5-7 hours
post dosing(46). These data indicate that prolonged vasodilation by
certain agents will contribute to vascular injury.

#### Flow-mediated coronary arterial lesion in dogs. {#flow-mediated-coronary-arterial-lesion-in-dogs. .unnumbered}

Several vasoactive agents that lower blood pressure and increase heart
rate induce regional, right coronary arterial damage (24, 58). The basis
for this selective coronary arterial lesion in dogs may be related to
localized increases in coronary blood flow because under normal
physiological conditions, localized control and regulation of coronary
blood flow is mediated by endogenous adenosine in response to increased
oxygen demand (3, 7, 9, 10). It is therefore not surprising that
pharmacologic mimicry of adenosine (i.e., adenosine agonists) by
compounds such as CI 914, CI 947 and N- (2,2-Diphenylethyl) adenosine
(DPEA) all induce coronary arterial lesions in dogs and increase
coronary arterial blood flow (4, 61, 69, 92). Therefore, it is now well
accepted that administration of adenosine (A1) agonists as a
pharmacological class are associated with coronary arterial lesions in
dogs. A "class" effect for coronary arterial lesions has also been
ascribed to K+ channel openers (68) and ETRAs (3, 58) because these
agents cause profound increases in regional blood flow.

Several reports from studies in dogs indicate that extreme increases in
regional CBF flow precede coronary arterial damage. For example
minoxidil, a long lasting vasodilator, when given to dogs at cardiotoxic
doses induced a 6-10 fold increase in regional cardiac blood flow and
this sustained increase in flow resulted in damage to the coronary
vasculature (42, 66). Other structurally and diverse pharmacologic
agents such as SB 209670 (59), hydralazine (18), SK&F 94836 (35) and
milrinone (53) which are vasoactive have been reported to increase CBF
and induce coronary arterial lesions in dogs.

Other studies in dogs have shown medial hemorrhage and necrosis of
extramural coronary arteries of the right atria that were associated
with minor but sustained increases in heart rate (10-30 beats/min) and
slight decreases in mean arterial pressure (10-15 mm Hg) after infusion
of an endothelin receptor antagonist for 5 days (57). These changes were
further shown to be associated with six-fold increases in regional blood
flows and a disproportionate distribution of ET~B~ receptors in the
right coronary arteries of dogs (59).

#### Blood flow and Biomechanical Injury {#blood-flow-and-biomechanical-injury .unnumbered}

It has been postulated that mesenteric and coronary arterial lesions
develop because of marked vasodilation, increased blood flow, increased
shear (endothelial cells) and hoop stress (vessel wall). Hoop stress or
tension is the product of transluminal pressure and the vessel radius
divided by wall thickness. Increased shear stress leads to
inter-endothelial breaks gradual breakdown of vessel wall integrity,
breaks in the internal elastic lamina and hemorrhage (46, 66). It has
also been speculated that vasodilation changes normal laminar flow to
turbulent flow that imitates shear stress and this leads to a cascade of
effects in the vessel wall culminating in hemorrhage and necrosis. It is
not clear to what extent similar changes occur in humans given
pharmacologically-active doses of certain vasodilators. It is also not
clear that such changes in vessel wall tension in the absence of
concurrent receptor mediated cellular alterations would result in the
observed vessel injury.

### Toxicity following direct pharmacological and/or chemical perturbation.

The target cell of drug-induced vascular toxicity is presumed to be
endothelial and/or smooth muscle cell. Development of vascular lesions
has been linked to the pharmacologic effect of the drug because the
pharmacologic target is often located on these cells. For example ETRAs
as a class cause arterial lesions in dogs because the drug target,
endothelin receptors, are located on endothelial and smooth muscle cells
(19). ET receptors on EC and SMC regulate vascular tone through opposing
vasoconstrictor and vasodilator effects (19, 20). Therefore, an upset of
this delicate balance resulting in loss of vasoconstriction or
vasodilation might lead to vascular damage. For example, it was shown
that the disproportionate distribution of vasoconstrictive receptors
which when antagonized within the right atrium and right coronary
arteries predisposes these sites to pharmacological vasodilation
resulting in six-fold increases in regional blood flow, but minimal
changes in heart rate and blood pressure (59). A similar observation has
been reported in the rat for DA~1~ receptors that are the target for
fenoldopam (48). Other pharmacological targets associated with vascular
injury that are located on EC and SMC cell include adenosine receptors,
K+ channels, and the cGMP-inhibitable isoenzyme of phosphodiesterase
(47). Vascular toxicity then may result from pharmacological interaction
with the molecular target that initiates a series of interactive
cascades among cellular and non-cellular components leading to eventual
damage. Contributing to toxicity may be the sustained alterations by
these agents of EC components normally responsible for targeting
interactions with the immune system.

Allylamine and β-Amino-proprionitrile (β--APN) are chemical substances
that are direct acting vascular toxicants (13). Allylamine causes medial
hypertrophy and sub-intimal proliferation and β--APN causes aneurysms of
the aorta, coronary and mesenteric vasculature in man (13). Based on the
different morphologic presentations of vascular injury induced by these
agents, it is reasonable to assume that the pathophysiology is quite
different from drug-induced lesions that tend to present a similar
morphologic picture.

### Immune-mediated Injury

Although the precise mechanisms by which immune cells mediate injury to
endothelial cells (EC) and smooth muscle cells is not clear, there is
evidence that drugs and biologics can activate the immune system which
in turn can cause EC injury. Moreover, drugs may modulate the EC
directly in such a way as to trigger an inflammatory response and an
immune attack directed against EC and smooth muscle. While the immune
effectors mediating acute drug-induced vascular injury have not been
extensively identified, several types of vasculitis as seen in chronic
autoimmune diseases and a number of infections have been well
characterized. Thus, the latter may serve as a model to address the
possible mechanisms involved in acute and chronic phases of drug-induced
vascular injury. Such disease-related vascular injuries have also been
discussed below to provide additional clues on how drugs may induce
immune-mediated vascular injury. The immune-mediated vasculitis can
involve multiple effector mechanisms as described below:

#### Mediators of immediate hypersensitivity {#mediators-of-immediate-hypersensitivity .unnumbered}

[Neutrophils]{.underline}. Neutrophils have been widely associated with
vascular injury. Chemotactic agents including complement components,
factors from fibrinolysis, activate them and kinin systems and
chemokines produced by endothelial cells and by other leukocytes all
activate neutrophils. Chemotactic stimuli facilitate their binding to
the endothelial cells. Neutrophils carry a variety of granules
containing hydrolases, myeloperoxidase, proteinases, and muramidase. In
certain forms of vascular injury, antibodies against myeloperoxidase
have been reported. Immune complexes can bind Fc receptors on
neutrophils and activate them to release enzymes which could cause
endothelial cell injury. Cytokines at the inflammatory site may
contribute significantly to the generation of reactive oxygen species
(ROS), particularly by PMN. The vascular endothelium is a major target
of oxidant stress, playing a critical role in the pathophysiology of
several vascular diseases and disorders. Specifically, oxidant stress
increases vascular endothelial permeability and promotes leukocyte
adhesion, which trigger alterations in endothelial signal transduction
and redox-regulated transcription factors (60). Histopathological
analysis of drug-induced vascular injury in the skin has shown two
distinct patterns (14). The first type consists mainly of neutrophils
and the second, predominantly mononuclear cells.

[Mast Cells]{.underline}. Mast cells have been well characterized for
their role in induction of allergies. Although their precise role in
endothelial cell injury is not clear, several recent studies suggest
that they may play a key role in endothelial cell functions and injury.
For example, mouse or human mast cells can produce and secrete vascular
permeability factor/vascular endothelial cell growth factor (VPF/VEGF)
which can potently enhance vascular permeability and induce
proliferation of vascular endothelial cells (12). Mast cells play a
critical role in regulating the expression of EC adhesion molecules,
ICAM-1 and VCAM-1, and thereby enhance leukocyte-mediated inflammatory
response (64). In mercuric chloride-induced vasculitis in rats, the role
of mast cells was implicated (51). In SK&F 95654 induced mesenteric
vasculitis in rats, enhanced local degranulation of mast cells was noted
(103).

#### Endothelial Cells {#endothelial-cells .unnumbered}

Endothelial cell activation has been implicated as an important event in
the induction of vasculitis and vascular leak syndrome. Endothelial cell
activation is considered as an immunological activation (11). Activated
endothelial cells can function as antigen-presenting cells and
participate in T cell mediated immune reactions (77). Activated
endothelial cells up-regulate expression of major histocompatibility
complex (MHC)-encoded molecules (77). MHC class I antigens on the
activated endothelial cells interact with CD8 T lymphocytes, while
endothelial MHC class II antigens interact with CD4 T lymphocytes in
cell-mediated immune responses (77). Furthermore, activated endothelial
cells express a number of immunologically relevant surface molecules,
such as adhesion molecules of the immunoglobulin gene superfamily. In
SK&F 95654 induced mesenteric vasculitis in rats, evidence for early
endothelial cell activation was reported (103). Increased numbers of
circulating endothelial cells have been detected during vascular injury
such as during myocardial infarction and endotoxinemia (30, 70). Thus,
it is possible that circulating endothelial cells may serve as a
biomarker for vascular injury.

#### Adhesion Molecules {#adhesion-molecules .unnumbered}

Several endothelial cell adhesion molecules belonging to the Ig
superfamily play a critical role in the interaction between EC and
leukocytes. These include ICAM-1, ICAM-2, VCAM-1, and MAdCAM. These
molecules are either expressed or inducible in EC. In addition,
integrins present on leukocytes are involved in adhesion to EC. Also,
E-selectin expressed on EC binds to the carbohydrate ligands on
leukocytes. Adhesion molecules play a critical role in the recruitment
of leukocytes in many forms of vascular injury. Increased expression of
ICAM-1 has been reported in vasculitic lesions involving nerve and
muscle as well as in vessels of SLE patients (63, 73). Also, in patients
with diffuse vasculitis, the skin vessels increased expression of
E-selectin and ICAM-1 (52). The levels of soluble ICAM-1 have been shown
to be higher in individuals who develop atherosclerosis there by
suggesting that sICAM-1 may serve as a biomarker for such vascular
lesions. ICAM-1 immunostaining on endothelial cells has been shown to be
enhanced in the mesentery of rats exposed to SK&F 95654 (103).

#### Autoantibodies {#autoantibodies .unnumbered}

Several drugs can cause vascular injury associated with anti-neutrophil
cytoplasmic autoantibodies (ANCA) (65). These patients produce Abs
primarily against myeloperoxidase or proteinase 3 (15, 56). Although it
is not clear that these antibodies are directly pathogenic, the levels
of ANCA may serve as a marker for disease activity and response to drug
withdrawal (65). There is strong evidence to suggest that hydralazine
and propyl thiouracil can cause ANCA-positive drug-induced vascular
injury (65). In addition to ANCA, anti-EC Abs (AECA) has also been
detected in a variety of vascular diseases. AECA may upregulate
expression of adhesion molecules such as E- selectin, ICAM-1 as well as
cytokines and chemokines by EC, which in turn may recruit leukocytes
(78).

#### T Cells {#t-cells .unnumbered}

Rats treated with mercuric chloride develop necrotizing vasculitis which
occurs in two phases: an early and a late phase. In the latter phase, T
cells seem to play an important role inasmuch as treatment with
monoclonal antiboidies to inhibit T cell function completely prevents
the development of late vasculitis (51). Furthermore, infiltration of T
cells into vascular lesions and elevated soluble IL-2 receptors in the
serum have been reported in Kawasaki disease (95). In Takayasu
arteritis, which exhibits vasculitis that involves the aorta and
pulmonary arteries, the infiltrating cells consist of cytotoxic T cells
and NK cells. They injure the vascular cells by producing perforin (85).
Vascular injury caused by cytolytic T cells and NK cells has been well
documented with the use of IL-2 and other cytokine therapies including
GM-CSF, IL-1 and IL-4 (97). IL-2 was shown to activate T cells to
express high levels of CD44, which interacts with EC and induces EC
injury by producing Fas ligand and perforin (79, 80). Activated
cytolytic T cells and NK cells express Fas ligand which in turn can
induce significant levels of apoptosis in EC (44). In giant cell
arteritis, the vascular injury appears to depend on T cells that produce
IFN-which activates macrophages (101). In a patient with acute drug
induced cutaneous vasculitis, a 100-fold increase in plasma
concentrations of IFN- was noted thereby suggesting that IFN- may
up-regulate adhesion molecule expression on EC and activation of T cells
(87).

#### Macrophages {#macrophages .unnumbered}

Activated macrophages are known to participate in the process of
instability and rupture of atherosclerotic plaque (100). In giant cell
arteritis, the mechanisms of injury have been mostly attributed to the
effector macrophages. Macrophages specialize in oxidative damage with
lipid peroxidation attacking smooth muscle cells and matrix components.
These macrophages also produce reactive oxygen intermediates which in
combination with nitrogen intermediates, cause protein nitration of
endothelial cells (100). In Kawasaki disease, activation of cytokines
produced by macrophages elicit proinflammatory and prothrombotic
responses in endothelial cells (43). In this disease, macrophage colony
stimulating factor has been shown to play a critical role in the
pathogenesis and can be used as an indicator for the risks of valvulitis
and coronary arteritis (43).

## Research Opportunities

1.  Identification and development of mechanistically based novel
    biomarkers of drug-induced vascular injury that targets both the
    vascular cells and immune cells.

2.  Direct measurement of early and sustained alterations in regional BF
    using flow in affected areas of vascular injury probes, radioactive
    or color-coated fluorescent microspheres (31)

3.  Measurement of the coronary arterial diameter by echocardiography
    (93).

4.  Evaluation of endothelial nitric oxide synthetase (eNOS) because it
    is elevated by increased shear stress. It has been suggested that
    increased shear elevates eNOS which is co localized with caveolae
    and this coupling transducer signals into EC as well as SMC
    cells (6) in the vessel wall.

5.  Develop methodologies (*in vitro* followed by *in vivo* studies) for
    direct measurement of shear stress or biochemical assays to
    determine eNOS or caveolin-1 concentrations that may be elevated
    because of increased shear.

6.  Electron microscopy to evaluate the status of EC and SMC caveolae
    because disruption of this cellular organelle is associated with
    vascular pathology in caveolin-1 gene disruption in mice (26).

7.  Immunohistochemistry, Western Blotting, Laser capture dissection and
    microarray technology to evaluate gene and protein changes in target
    cells i.e., EC and SMC.

8.  For agents that are potentially direct vascular toxicants employing
    cytotoxicity assays with cultured EC and SMC at relevant
    concentrations that may be achieved in *vivo* toxicology studies.

9.  Identification of the mechanisms by which immune cells are activated
    directly by the drugs, which in turn may contribute to EC injury.
    This involves identification of the nature of effector cells (e.g.,
    neutrophils, lymphocytes, monocytes, macrophages, basophils,
    eosinophils, mast cells) adhesion molecules that are upregulated on
    leukocytes, which facilitate the leukocyte-EC interactions, and the
    effector molecules produced by leukocytes involved in EC injury.

10. In instances where drugs modulate the ECs in a way that would
    secondarily trigger an immune attack, it is important to identify
    the nature of molecular and phenotypic changes occurring in EC that
    trigger inflammation and immune attack.

11. The studies on the role of immune components in EC injury can be
    facilitated by the use of pharmacologically immunocompromised,
    genetically immunodeficient animals, transgenic knockout animal
    models deficient in specific immune components, and knock-in animal
    models where the original strain has a pathologic defect that can be
    corrected by supplying the normal functional gene.

12. Laser capture to isolate EC or inflammatory cells and DNA microarray
    analysis may help in identifying specific pathways of immune cell
    and EC activation.

13. The mechanism of immune cell-mediated EC injury such as necrosis or
    apoptosis will be useful in identifying better biomarkers and to
    develop strategies to prevent EC injury.

14. Role of anti-neutrophil cytoplasmic autoantibodies (ANCA) in
    drug-induced vascular injury.

15. Analyze genetic risk factors, which may play a role in
    immune-mediated EC injury.

16. Quantitative imaging of gene induction in living animals.

## Gaps

1.  Correlate blood flow changes in the dog using the various methods
    and different pharmacological agents to clearly establish what
    magnitude of blood flow increase causes vascular lesions.

2.  Technology is limiting for measuring blood flow in rat. A
    reproducible and validated model is not available and so the
    increased blood flow hypothesis in the rat has not been
    appropriately tested.

3.  Measurement of shear stress and biomechanical effects has not been
    attempted in rats or dogs.

4.  No available data on the biochemical (signal transduction pathway)
    effects of these drugs on EC or smooth muscle cells in vivo or in
    vitro. This evaluation may yield potential soluble markers
    measurable in serum and/or plasma.

5.  Lack of hard scientific data that provides an understanding of the
    biomechanical forces interplay in the vascular wall during
    vasodilation that leads to subsequent injury.

6.  Do shear stress and/or exaggerated pharmacology and vasodilation
    cause apoptosis of EC and SMC in the vessel wall?

7.  Understanding and being able to separate primary vs secondary
    effects.

8.  Understanding the nature of leukocyte-EC cell interactions during
    drug-induced vascular injury.

9.  To identify biomarkers of inflammation as they relate to
    drug-induced vascular injury.

## Summary

It is clear that a more complete understanding of the mechanisms of
drug-induced vascular injury will accelerate development of solutions to
managing risk. Directed approaches to understand mechanisms of vascular
injury will provide a link between structural damage and derangement of
specific cardiovascular functions. Testing of the prevailing hypotheses
as an initial approach in a decision tree paradigm will provide focus
and direction to the investigative activities. Secondly, unraveling the
mechanism of toxicity will ultimately lead to identification of
mechanistically relevant biomarkers because the investigative studies
will link the deranged cardiovascular alterations to cellular,
biochemical and molecular events. The ultimate product of this effort
then would be identification of mechanistically linked, relevant and
bridging biomarkers of vascular toxicity that has both nonclinical and
clinical application. Employment of new technologies such as proteomics,
metabonomics and transcriptomics should be utilized as appropriate in
these scientific investigations. Use of specific gene-targeted
transgenic and knock-out and knock-in animals should provide useful
information on the precise role of immune cells in drug-induced vascular
injury. It is also clear that a balance in the interaction between
leukocytes and endothelial cells is critical for immune system and
vascular homeostasis. In contrast, perturbations in this balance can
contribute to vascular and immune system anomalies. Thus, understanding
the effect of drugs on EC and immune cells would lead to development of
better models to study vascular injury and further help identify
biological markers that can evaluate and predict drug-induced vascular
injury.

# BIOMARKERS OF VASCULAR INJURY

## Introduction: Why Vascular Injury Biomarkers?

Data from animal toxicology studies are generally needed from drug
developers to demonstrate for regulatory agencies a safe strategy for
initiating human clinical trials**.** When drug-induced toxicities, such
as vasculitis (or vascular injury), occur in animals, reasonable
assurances for safety are needed from animal studies when appropriate
clinical monitoring is not feasible. Among the many goals for performing
animal toxicology studies are: 1) an identification of all dose-limiting
and associated toxicities and their reversibility, and identification of
the dose-exposure and dose-response relationships seen up to maximally
tolerated doses, 2) an identification of a safe starting dose for human
trials, and 3) a definition of an appropriately safe patient monitoring
strategy for clinical trials. Data from animal studies are best applied
to define the relationship between dose, drug exposure, therapeutic
effect, and dose-limiting toxicity in order to evaluate appropriateness
for clinical trials. In such animal studies involving extensive
histopathological evaluations, however, the use of serum or plasma
biomarkers is often limited, in routine analyses, as described by a
working group of clinical pathologists in 1996 (99). This working group
has recommended routine application of a core set of clinical endpoints
for routine toxicity screening. These endpoints, together with
hematology data, serve as reporters of diminished kidney function (e.g.,
BUN, creatinine), altered liver integrity (e.g., ALT, AST), altered
general homeostasis (e.g., electrolytes, pH), or tissue response to
injury (e.g., alterations in circulating cell populations). These same
clinical endpoints have been used for routine monitoring for well over
25 years despite great strides in biological research over this same
time frame. The integrity of most tissues is either not being monitored
or only monitored on a limited basis and clearly vascular injury is not
covered.

New chemical entities from several pharmacologic classes have been
developed that present with histopathological evidence of vascular
injury that is clearly associated with drug administration in a dose and
time dependent manner. Many of the earlier studies that have been
published in the peer-reviewed literature describing such findings, were
seen with agents at doses that produced dramatic effects on vasomotor
tone, blood pressure and heart rate. A reasonable level of assurance was
felt that if higher doses were avoided and cardiovascular parameters
were monitored in dose escalation clinical trials, the vascular injury
could be avoided in the clinic. However, many of the agents under
development more recently have been associated with histopathological
evidence of vascular injury in animal toxicology studies, without any
easily measureable effects on vascular tone, blood pressure, or heart
rate. In addition, in some cases the therapeutic index is not broad.
\[The therapeutic index is defined as the multiple between the exposure
achieved at the highest dose at which no dose limiting toxicity is
discovered, as compared to the exposure achieved at the lowest dose
achieving therapeutic effect in the same species.\] The lack of a
practical clinical monitoring strategy often results in the sponsor and
regulator sorting through speculative discussions surrounding relevance
of animal findings to humans, and the inaccuracies of any defined
exposure-response relationships during the proposed ensuing clinical
phases of development. There is growing concern and recognition of the
role of vascular injury and vascular inflammation as a risk for
cardiovascular disease, myocardial infarction, and stroke (75). There
are also experiences indicating that toxicities can sometimes be species
specific and irrelevant to humans. There is a need and desire for the
identification and validation of mechanism-based easily accessible
biomarkers that can be measured in animal toxicology studies and
integrated with histopathology studies to demonstrate their ability to
herald the early onset and monitor progression or reversibility of
drug-induced vascular injury across pharmacologic classes and across
species, including humans.

## Scope of the problem and paths forward

The pathogenesis of human idiopathic vasculitides are poorly understood
and definitive and distinctive diagnoses of each presentation can be
difficult. The underlying mechanisms, the earliest initiating events,
the role and interplay of specific immune cell components involved, the
vessels targeted, the progression of early disease to later forms, the
availability of diagnostic biomarkers are all important areas under
active investigation. Currently recognized biomarkers for human
vasculitides are of limited value, tend to be focused on immune
manifestations of the disease and may reflect late stages of disease.
For example, anti-neutrophil cytoplasmic antibodies to neutrophil
myeloperoxidase (pANCA) and to neutrophil anti-proteinase 3 (cANCA) are
known to be late-appearing and are not present in all forms of the
clinical vasculitides. Furthermore, these and other markers which are
presently the subject of ongoing clinical research are of unknown and
largely untested value to animal toxicology studies. However, in the
case of atherosclerosis, another systemic inflammatory disease, several
prospective clinical studies have shown that plasma levels of
high-sensitivity C-reactive protein (hsCRP), an acute phase reactant, is
a strong predictor of future myocardial infarction and stroke in
otherwise healthy men and women (81). Measurement of hsCRP in humans may
soon become routine as a screen for risk of cardiovascular events. For
the many examples of drug-induced vascular injury that have been noted
in animal models, there are likely to be differences among the models
and a blurring of boundaries across mechanisms. It will be important to
carefully characterize the presentation of histopathology by identifying
the vessels and tissues affected and to link this data with biomarker
alterations, and to understand pathogenesis, to understand the role of
specific immune components in initiation and progression of disease. For
pragmatic solutions, the identification of practical easily accessible
diagnostic interspecies biomarkers is of prime importance. Biomarkers
useful to drug and biologic development would ideally: be useful for
defining specific pathogenic mechanisms, be easily accessible, be
sensitive and specific to appear with the earliest manifestations of
vascular injury, define the site and type of vessel affected, track with
disease severity and regress as disease mitigates, be measurable with
reagents that identify regions of proteins conserved across species
including humans. It is also recognized that biomarkers whose
performance attributes may fall short of these ideals, may nevertheless
have some value in specific contexts. It is likely that because
pathogenic mechanisms will be different across pharmacologic classes and
even across species within a class, and that the presentation of
pathology will vary by vessel and tissues affected, panels of biomarkers
may be necessary to help detect all forms of vascular injury and
distinguish mechanisms.

Genomic, proteomic, and metabonomic technologies are expected to assist
with questions of interspecies relevance by helping to define pathogenic
mechanisms and by identifying additional monitorable interspecies
biomarkers. Such approaches are both hypothesis-generating and
hypothesis-directed. When careful attention is paid to study design and
pivotal comparators are built onto careful investigative observation,
the distinction between hypothesis generation and investigation is
subtle and the value of these technologies especially in biomarker
discovery should not be underestimated. Careful analyses of genomic and
proteomic biomarker alterations at sites of tissue injury could
elucidate pathogenic mechanisms. Investigations of altered protein and
gene expression in circulating immune cells could serve a practical role
in identifying accessible and monitorable biomarkers. Investigations of
serum and urine components using proteomic and metabonomic technologies
are already beginning to yield useful results. Expansion of such
approaches is expected. In addition to such broad based biomarker
discovery approaches, the systematic measurements of alterations in
specific endpoints as tabulated below are described based on an
evaluation of the published literature. These endpoints are hypothesized
to be associated with drug-induced vascular injury and are furthermore
hypothesized to add value to investigations of drug-induced vascular
injury in drug developmental regulatory studies. To further investigate
the value of these potential biomarkers, collaborative approaches are
envisioned with the design of common protocols and appropriate controls,
technology experts contributing common assay reagents, and a strategy
developed toward both analytical and biological validation with a bridge
toward clinical utility optimized. The following tabulation of potential
biomarkers has been derived based on a recognition of a common
association of manifestations of vascular injury with either 1)
inflammation, inflammatory mediators, and the appearance of certain
acute phase reactants; 2) the appearance of adhesion molecules and
integrins; 3) the observation of excessive endothelial and smooth muscle
cell activation and injury including both necrotic and apoptotic forms;
4) the observed and hypothesized involvement of diverse immune cell
components including neutrophils, basophils, mast cells, and
T-lymphocytes. An attempt has been made to develop some consensus toward
prioritizing the investigations of the associations of these endpoints
with drug-induced vascular injury.

## Potential Biomarkers

<table>
<colgroup>
<col style="width: 20%" />
<col style="width: 9%" />
<col style="width: 7%" />
<col style="width: 0%" />
<col style="width: 7%" />
<col style="width: 0%" />
<col style="width: 40%" />
<col style="width: 1%" />
<col style="width: 5%" />
<col style="width: 5%" />
</colgroup>
<thead>
<tr class="header">
<th colspan="10"><strong>VASCULAR INJURY EXPERT WORKING GROUP-POTENTIAL
BIOMARKERS</strong></th>
</tr>
</thead>
<tbody>
<tr class="odd">
<td><strong>Rationale: Biomarker Group/Class Section
Author</strong></td>
<td><strong>Biomarker</strong></td>
<td colspan="2"><strong>Contact</strong></td>
<td colspan="2"><strong>Reagent-Test Availability</strong></td>
<td><strong>Key points, references, confounders and other Issues
(species relevancy, timing, specificity)</strong></td>
<td colspan="2"><p><strong>Current Usefulness</strong></p>
<p><strong>(A =high B=mod)</strong></p></td>
<td><strong>Future Promise RD Status (A=high B=mod)</strong></td>
</tr>
<tr class="even">
<td><strong>Endothelial Cell Targets (Thomas)</strong></td>
<td>sE-selectin (CD62E)</td>
<td colspan="2"><p>Kerns</p>
<p>Zhang</p></td>
<td colspan="2">Rat - N Dog - N Human - Y</td>
<td><ul>
<li><p>expressed selectively on endothelial cells following activation
by TNF, IL-1 and LPS</p></li>
<li><p>ligands include: sialyl-Lewis (sCD15), P-selectin glycoprotein
ligand-1, cutaneous lymphocyte antigen</p></li>
<li><p>involved in leukocyte tethering and rolling velocity reduction
during the initial stages of inflammation and following endothelial
activation</p></li>
<li><p>is shed from the endothelium and circulates as sCD62E in several
inflammatory diseases that affect the endothelium, including diabetes,
atherosclerosis, smoking etc</p></li>
<li><p>in healthy individuals, the levels of circulating sCD62E are very
low</p></li>
<li><p>potential marker with high sensitivity and high specificity in
healthy volunteers and high sensitivity and moderate to low specificity
in selected patient populations</p></li>
<li><p>human tests are available</p></li>
<li><p>no rat reagents or test available</p></li>
<li><p>proposal submitted for review to set up and validate rat ELISA
test system. ~10K; per sample cost will depend on volume</p></li>
<li><p>clinical site in Ireland is willing to collaborate for Fenoldopan
study</p></li>
<li><p>May serve as an early biomarker for vasculitis. The maximal
transcript level of E-selectin at 3-6 hr in a murine model (Fries JWU,
Williams AJ, Atkins RC, Newman W, Lipscomb MF, and Collins T: Expression
of VCAM-1 and E-selectin in an <em>in vivo</em> model of endothelial
activation. Am J Pathol 143:725-737, 1993)</p></li>
<li><p>High degree of structural and functional homology of E-selectin
among human, mouse, and rabbit (Becker-Andre M, van Huijiduinen RH,
Losberger C, Whelan J, Delamarter JF: Murine endothelial
leukocyte-adhesion molecule-1 is a close structural and functional
homologue of the human protein. Eur J Biochem 206:401, 1992) (Weller A,
Isenmann S, Vestweber D: Cloning of the mouse endothelial selectins:
Expression of both E- and P-selectin is inducible by tumor necrosis
factor. J Bio Chem 267:15176, 1992)</p></li>
<li><p>Significant increase of soluble levels of E-selectin in patients
with a variety of vasculitides (≈ 1-3-fold increase above control
subjects)</p></li>
</ul></td>
<td colspan="2"></td>
<td>A</td>
</tr>
<tr class="odd">
<td><strong>Rationale: Biomarker Group/Class Section
Author</strong></td>
<td><strong>Biomarker</strong></td>
<td colspan="2"><strong>Contact</strong></td>
<td colspan="2"><strong>Reagent-Test Availability</strong></td>
<td><strong>Key points, references, confounders and other Issues
(species relevancy, timing, specificity)</strong></td>
<td colspan="2"><p><strong>Current Usefulness</strong></p>
<p><strong>(A =high B=mod)</strong></p></td>
<td><strong>Future Promise RD Status (A=high B=mod)</strong></td>
</tr>
<tr class="even">
<td>Endothelial cells comprise the sole cell type lining the lumen of
all vasculature in the body with approximately 10<sup>13</sup> cells
covering 7 m<sup>2</sup> in the average adult human (Holash, J et. al.,
Science 284:1994-1998 (1999)). They regulate nutrient and blood
component traffic to all tissues in the body, and are also intimately
involved in control of blood flow, hemostasis and inflammation. As such,
endothelial cells and their secreted and/or synthesized proteins should
be considered prime targets for providing potential biomarkers of
vascular injury. Circulating endothelial cells shed from the vascular
wall have been documented after vascular damage due to multiple
etiologies (reviewed in Dignat-George, F and J. Sampol, European J
Haematol 65:215-220 (2000). Of the secreted endothelial cell proteins,
von Willebrand Factor (vWF) has received the most attention as a
potential biomarker of vascular damage (Lip, G.Y.H. and A. Blann,
Cardiovascular Research 34:255-265 (1997)).</td>
<td>Circulating Endothelial Cells (CEC)</td>
<td colspan="2">Thomas</td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td><ul>
<li><p>may need to use a combination of surface markers to identify
including CD31, acetylated lipids (DiI-Ac-LDL)</p></li>
<li><p>shown to increase in number after varied causes of vascular
damage in people</p></li>
<li><p>potential tail vein blood-drawing high background levels in
rats</p></li>
</ul></td>
<td colspan="2"></td>
<td>A</td>
</tr>
<tr class="odd">
<td></td>
<td>vWF</td>
<td colspan="2">Thomas</td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td><ul>
<li><p>Elevation of plasma and serum protein after varied causes of
vascular damage in people, and after drug-induced vascular injury in
rats( although increase is short-lived)</p></li>
<li><p>sequential blood drawing artifacts</p></li>
<li><p>vWF elevated in smokers</p></li>
</ul></td>
<td colspan="2">B</td>
<td></td>
</tr>
<tr class="even">
<td></td>
<td>vWF pre-protein</td>
<td colspan="2">Louden</td>
<td colspan="2">Rat - N Dog - N Human - Y</td>
<td><ul>
<li><p>Sensitive marker acute and progressive injury; not found in
normal plasma, rapid turnover and short half-life, releases as a 1:1
ratio with mature protein</p></li>
<li><p>no extensive human experience, baboon only animal species with
data.</p></li>
<li><p>Refs:Thrombosis and Haemostasis 80:, 1002-1007; 1998 vWF
pro-peptide</p></li>
</ul></td>
<td colspan="2"></td>
<td>B</td>
</tr>
<tr class="odd">
<td></td>
<td>endothelial microparticles (EMPs)</td>
<td colspan="2">Zhang</td>
<td colspan="2">Rat - ? Dog - N Human - Y</td>
<td><ul>
<li><p>A marker of endothelial cell activation in thrombotic
thrombocytopeienic purpura</p></li>
<li><p>The number of EMPs can be measured by monoclonal antibodies CD31
and CD51 using flow cytometry</p></li>
<li><p>Elevated EMPs in the plasma correlate to increased expression of
ICAM-1 and VCAM-1</p></li>
<li><p>CD31 (also called PECAM-1) is expressed on endothelial cells,
platelets, and megakaryocytes. It plays a role in the interaction of the
endothelium and platelets</p></li>
<li><p>CD51/CD61 (also called 23 integrin) is an endothelial cell
adhesion molecule, which is mainly expressed on endothelial cells but is
also expressed on leukocytes and macrophages. It plays a role in the
interaction of endothelial cells and extracellular matrix.</p></li>
<li><p>Refs: Jimenez JJ, Jy W, Mauro LM, Horstman LL, and Ahn YS (2001).
Elevated endothelial microparticles in thrombotic thrombocytopenic
purpura: Findings from brain and renal microvascular cell culture in
patients with active disease. Br J Haematol. 112:81-90. Ferrer L,
Fondevila D, Rabanal RM, and Vilafranca M (1995). Immunohistochemical
detection of CD31 antigen in normal and neoplastic canine endothelial
cells. J Comp Pathol. 112:319-326. (related to CD31and CD51on
endothelial cells and platelets)</p></li>
</ul></td>
<td colspan="2"></td>
<td>B</td>
</tr>
<tr class="even">
<td><strong>Smooth Muscle Cell Targets</strong></td>
<td>caveolin</td>
<td colspan="2">Louden</td>
<td colspan="2">Rat - Dog - Human -</td>
<td><ul>
<li><p>Caveolin-1, plasmalemmal invaginated vesicle, key role in cell
signaling pathway</p></li>
<li><p>expressed on EC, SMC and fibroblasts; loss impairs nitric oxide
and calcium signaling causing aberrations in endothelium-dependent
relaxation, contractility and maintenance of myogenic tone. See immuno
histochemistry slide for caveolin-1</p></li>
<li><p>expressed on EC, SMC and fibroblasts; loss impairs nitric oxide
and calcium signaling causing aberrations in endothelium-dependent
relaxation, contractility and maintenance of myogenic tone. See
immunohistochemistry slide for caveolin-1</p></li>
<li><p>Refs: Thrombosis and Hemostasis, 77: 387-393,</p></li>
</ul></td>
<td colspan="2"></td>
<td>B</td>
</tr>
<tr class="odd">
<td>Smooth muscle surrounding vessels is often seen to be disrupted
during vascular injury; smooth muscle may be disrupted by hemodynamic
factors, or may be a direct target of cell injury</td>
<td>smooth muscle alpha actin</td>
<td colspan="2">Thomas</td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td><ul>
<li><p>Smooth muscle alpha actin expressed in vascular smooth
muscle</p></li>
<li><p>Antibody available which cross reacts across mammals</p></li>
<li><p>unknown whether increases in circulation after vascular
injury</p></li>
</ul></td>
<td colspan="2"></td>
<td>B</td>
</tr>
<tr class="even">
<td><strong>White Blood Cell Targets (Burchiel)</strong></td>
<td>Neutrophils</td>
<td colspan="2">Burchiel</td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td><ul>
<li><p>Neutrophils and monocytes are oftentimes seen in areas of
vascular damage</p></li>
<li><p>unclear whether inflammatory cells are the cause or result of
vascular damage</p></li>
</ul></td>
<td colspan="2">A</td>
<td></td>
</tr>
<tr class="odd">
<td>WBC infiltrates are oftentimes seen in histopathologic lesions
associated with vascular damage which is termed "vasculitis" - unclear
whether inflammatory cells are the cause or result of vascular
damage</td>
<td>Mast Cells and Basophils</td>
<td colspan="2"><p>Burchiel</p>
<p>Zhang</p></td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td><ul>
<li><p>Recent studies by Zhang, Hermann, and Sistare at CDER demonstrate
that mast cells are oftentimes found associated with lesions in SHR
Wistar-Kyoto rats; due to the rapid onset of vascular lesions in this
species, a possible mechanism of vascular injury may relate to
"pseudoallergy"</p></li>
<li><p>Recent studies by Zhang, Herman and Sistare at CDER demonstrated
that increased mast cells are oftentimes associated with mesenteric
vascular injuries in SD, SHR, and WKY rats treated with the PDE III
inhibitor, SK&amp;F 95654; the possible release of inflammatory
mediators and cytokines from mast cell degranulation is suggested (Zhang
J, Herman EH, Knapton A, Chadwick DP, Whitehurst VE, Korerner JE,
Papoian T, Ferrans V, and Sistare FD: SK&amp;F 95654-induced acute
cardiovascular toxicity in Sprague-Dawley rats: Histopathologic,
electron microscopic, and immunohistochemical studies. Toxicol Pathol
30:28-40, 2002)</p></li>
</ul></td>
<td colspan="2"></td>
<td>B</td>
</tr>
<tr class="even">
<td><strong>Acute Phase Reactant Proteins (Blanchard)</strong></td>
<td>C-reactive protein (CRP)</td>
<td>Blanchard</td>
<td colspan="2">Rat - Y dog - Y Human - Y</td>
<td colspan="3"><ul>
<li><p>C-reactive protein is relatively conserved in its response
amongst species (rat, dog and man). CRP is believed to bind bacterial
and fungal cell walls, resulting in opsonized particles and serving as a
complement cascade attractant; CRP is elevated in animals (rats and
dogs) with vascular lesions</p></li>
</ul></td>
<td>A (for relevant species)</td>
<td></td>
</tr>
<tr class="odd">
<td>An acute phase inflammatory response, often elicited by endogenous
pyrogens such as IL-1, IL-6 or TNFa, involves the secretion of a variety
of liver proteins into the blood (Loeb and Quimby 1999). Not
unexpectedly, this response varies amongst species and is a general
marker of inflammation not specific to vasculitis. Two such acute phase
reactants, namely c-reactive protein and serum amyloid A, have recently
been reported to coincide with drug-induced vasculitis in the dog (Zhang
2002)</td>
<td>Serum amyloid A</td>
<td>Blanchard</td>
<td colspan="2">Rat - N Dog - Y Human - Y</td>
<td colspan="3"><ul>
<li><p>Serum amyloid A appears to respond as an acute phase protein in
man and dog, but may not be an appropriate marker in rat</p></li>
<li><p>The function of serum amyloid A is not completely understood but
may play a role in lipid metabolism during inflammation.</p></li>
</ul></td>
<td>A (for relevant species)</td>
<td></td>
</tr>
<tr class="even">
<td></td>
<td>Haptoglobin</td>
<td>Blanchard</td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td colspan="3"><ul>
<li><p>Haptoglobin is a mucoprotein that traps hemoglobin from lysed red
blood cells; it has been reported to be elevated in inflammatory
disorders. Zhang J et al., Acute Phase Proteins (APPs) as biomarkers of
host-response to drug-induced vasculitis in rats and dogs. 2002 FDA
Scientific Forum, Abstract T3</p></li>
<li><p>haptoglobin has been found to be elevated in dogs with vascular
lesions</p></li>
<li><p>Ref: Zhang J, Herman E, Holt G, Blanchard K, Ratajczak H, Knapton
A and Sistare F: Acute phase proteins (APPs) as biomarkers of
host-response to drug-induced vasculitic in rats and dogs. Toxicological
Sciences. 66 (1-S):15, 2002</p></li>
</ul></td>
<td>A (for relevant species)</td>
<td></td>
</tr>
<tr class="odd">
<td></td>
<td>Fibrinogen</td>
<td>Zhang</td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td colspan="3"><ul>
<li><p>An acute phase protein related to the release IL-6 in
vasculitis</p></li>
<li><p>-fibrinogen is increased to 4.8 fold 4 hr after the
administration of IL-6 in male Wistar rats (Geiger T, Andus T, Klapproth
J, Hirano T, Kishimoto T, and Henrich PC (1988). Induction of rat
acute-phase proteins by interleukin 6 in vivo. Eur J Immunol
18:717-721)</p></li>
<li><p>Fibrinogen is increased to 3.8 fold after the administration of
IL-6 in human hepatocytes (Castell JV, Gomez-Lechon MJ, David M, Hirano
T, Kishimoto T, and Heinrich PC (1988). Recombinant human interleukin-6
(IL-6/BSF-2/HSF) regulates the synthesis of acute phase proteins in
human hepatocytes. FEBS Lett. 232:347-350)</p></li>
</ul></td>
<td>B</td>
<td></td>
</tr>
<tr class="even">
<td></td>
<td>2-macroglobulin</td>
<td>Blanchard</td>
<td colspan="2">Rat - Y Dog - N Human - N</td>
<td colspan="3"><ul>
<li><p>this particular protein may be a respondent specifically in the
rat and is not relevant for dogs and humans</p></li>
<li><p>some data demonstrate that this marker is elevated in rats with
vascular lesions</p></li>
</ul></td>
<td>A (for relevant species)</td>
<td></td>
</tr>
<tr class="odd">
<td><strong>Inflammatory (Nagarkatti)</strong></td>
<td>IL-1</td>
<td>Nagarkatti</td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td colspan="3"><ul>
<li><p>Interleukin-1 (IL-1) is an important mediator of the immune
system, playing key role during infection, inflammation,
cell-differentiation, tissue remodelling, and apoptosis</p></li>
<li><p>It may act as an activator of cardiovascular cells inasmuch as
cells of the vessel wall and the heart can produce IL-1</p></li>
<li><p>IL-1 can be detected in the serum using ELISA; one plate costs
~$300.</p></li>
</ul></td>
<td>B</td>
<td></td>
</tr>
<tr class="even">
<td>Inflammatory cytokines and chemokines, much like acute phase
reactant proteins, are nonspecific indicators of inflammation; Pober JS,
Cotran RS. Cytokines and endothelial cell biology. Physiol Rev. 1990;
70: 427-51.</td>
<td>IL-6</td>
<td>Nagarkatti</td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td colspan="3"><ul>
<li><p>IL-6 is primarily produced by macrophages, TH2 cells and bone
marrow stromal cells</p></li>
<li><p>There are reports suggesting that in some types of vasculitis,
IL-6 may play a critical role</p></li>
<li><p>It induces synthesis of acute phase proteins in the
liver</p></li>
<li><p>IL-6 can be detected in the serum using ELISA; One plate costs
~$300</p></li>
<li><p>Ref: HogenEsch H, Snyder PW, Scott-Moncrieff JCR, Glickman LT,
and Felsburg PJ: Interleukin-6 activity in dogs with juvenile
polyarteritis syndrome: Effect of corticosteroids. Clin Immunol
Immunopathol. 77:107-110, 1995.</p></li>
</ul></td>
<td>B</td>
<td></td>
</tr>
<tr class="odd">
<td></td>
<td>TNF</td>
<td>Nagarkatti</td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td colspan="3"><ul>
<li><p>TNF is a major mediator of apoptosis as well as inflammation and
immunity</p></li>
<li><p>TNF is the prototypic proinflammatory cytokine and endothelial
cells are the principal cellular targets of its actions</p></li>
<li><p>It is found in the serum and can be detected using ELISA; one
plate costs ~$300.</p></li>
</ul></td>
<td>B</td>
<td></td>
</tr>
<tr class="even">
<td></td>
<td>PG's and LT's</td>
<td>Burchiel</td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td colspan="3"><ul>
<li><p>RIAs and ELISAs exist for a variety of prostaglandin (PGs) and
leukotrienes (LTs) and their major serum and urinary
metabolites</p></li>
<li><p>these are nonspecific indicators of inflammatory disease are of
unproven use in vasculitic</p></li>
</ul></td>
<td>B</td>
<td></td>
</tr>
<tr class="odd">
<td></td>
<td>Chemokines (MIP-1, MCP, IL-8)</td>
<td>Zhang</td>
<td colspan="2">Rat - Y Dog - Y/N Human - Y</td>
<td colspan="3"><p><u>IL-8 (chemokine)</u></p>
<ul>
<li><p>A marker of endothelial cell activation (Cockwell P, Tse WY, and
Savage COS (1997). Activation of endothelial cells in thrombosis and
vasculitis. Scan J Rhematol 26:145-150)</p></li>
<li><p>An activator of neutrophils (formerly known neutrophil activating
protein-1) and a potent neutrophil chemoattractant</p></li>
<li><p>It plays a role in the transendothelial migration of neutrophils,
particularly in post-capillary venules</p></li>
<li><p>It enhances the interaction of neutrophils with ICAM-1</p></li>
<li><p>Significant increase inn soluble levels of IL-8 in patients with
vasculitis (Tesar V, Jelinkova E, Masek Z, Jirsa M Jr, Zabka J,
Bartunkova J, Stejskalova A, Janatkova I, and Zima T (1998). Influence
of plasma exchange on serum levels of cytokines and adhesion molecules
in ANCAS-positive renal vasculitis. Blood Purif 16;72-80)</p></li>
</ul>
<blockquote>
<p><u>MCP-1, MIP-1, RANTES (chemokines)</u></p>
</blockquote>
<ul>
<li><p>MCP-1, MIP-1, and RANTES are synthesized and secrete by activated
endothelial cells</p></li>
<li><p>MCP-1 is an activator of monocytes and a monocyte
chemoattractant, thereby playing a role in the regulation of monocyte
extravasation</p></li>
<li><p>MIP-1 and RANTES are selectively chemoattractant for T
cells</p></li>
<li><p>Significant increase in MCP-1, MIP-1a, and RANTRES gene
expression levels in patients with vasculitis (Wong M, silverman ED, and
Fish EN (1997). Evidence for RANTES, monocyte chemotactic protein-1, and
macrophage inflammatory protein-1a expression in Kawasaki disease. J
Rheumatol 24:1179-1185)</p></li>
<li><p>various assays exist for chemokines and cytokines, including
those that are protein-based as well as mRNA-based. The useful of these
markers in vasculitis has not been established</p></li>
</ul></td>
<td>B</td>
<td></td>
</tr>
<tr class="even">
<td><strong>Adhesion Molecules (Zhang)</strong></td>
<td>sCD44-Hyaluronic Acid</td>
<td>Nagarkatti</td>
<td colspan="2">Rat - Y Dog - Y/N Human - Y</td>
<td colspan="3"><ul>
<li><p>Activation of T cells by Ag or stimulation of monocytes with
inflammatory cytokines induces CD44 to bind to hyaluronic acid (HA), an
adhesion event implicated in leukocyte-leukocyte and
leukocyte-endothelial cell interactions (Lesley J, Hyman R, Kincade PW.
CD44 and its interaction with extracellular matrix. Adv Immunol 1993;
54:271-335.)</p></li>
<li><p>CD44 expressed on activated T cells and NK cells is involved in
endothelial cell injury and vascular leak (Rafi-Janajreh AQ, Chen D,
Schmits R, Mak TW, Grayson RL, Sponenberg DP, Nagarkatti M, Nagarkatti
PS. Evidence for the involvement of CD44 in endothelial cell injury and
induction of vascular leak syndrome by IL-2. J Immunol. 1999; 163
:1619-27)</p></li>
<li><p>Also, activated endothelial cells also express CD44 (Griffioen
AW, Coenen MJ, Damen CA, Hellwig SM, van Weering DH, Vooys W, Blijham
GH, Groenewegen G. CD44 is involved in tumor angiogenesis; an activation
antigen on human endothelial cells. Blood. 1997;90:1150-9)</p></li>
<li><p>may be unique isoforms expression by endothelial cells; thus,
detection of soluble CD44 and HA in blood may constitute a useful
biomarker for vasculitis</p></li>
</ul></td>
<td></td>
<td>B</td>
</tr>
<tr class="odd">
<td></td>
<td>MAdCAM</td>
<td>Zhang</td>
<td colspan="2">Rat - Y Dog - Y/N Human - Y</td>
<td colspan="3"><ul>
<li><p>MAdCAM-1 is an endothelial cell adhesion molecule, which is
specifically expressed on the high endothelial venules in lymphoid
tissues</p></li>
<li><p>L-selectin and 47 integrin are receptors of MAdCAM-1</p></li>
<li><p>Involvement of L-selectin-mediated or 7 integrin lymphocyte
rolling</p></li>
<li><p>In rat, 4 integrins on the surface of neutrophils interact with
MAdCAM-1 on the endothelium, suggesting that rat neutrophils may use 4
integrins to mediate selective recruitment of neutrophils to sites of
inflammation in vivo ( Davenpeck KL, Sterbinsky SA, and Bochner BS
(1998)</p></li>
<li><p>Rat neutrophils express 4 and 1 integrins and bind to vascular
cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion
molecule-1 (MAdCAM-1). Blood. 91:2341-2346</p></li>
<li><p>unique to Peyer's patch endolthelial cells; Berg EL, McEvoy LM,
Berlin C, Bargatze RF, and Butcher EC (1993)</p></li>
<li><p>L-selectin-mediated lymphocyte rolling on MAdCAM-1. Nature.
366:695-698. Connor EM, Eppihimer MJ, Morise Z, Granger DN, and Grisham
MB (1999).</p></li>
<li><p>Expression of mucosal addressin cell adhesion molecule-1
(MAdCAM-1) in acute and chronic inflammation. J Leukoc Biol.
65:349-355.</p></li>
</ul></td>
<td></td>
<td>B</td>
</tr>
<tr class="even">
<td></td>
<td>ICAM-1</td>
<td>Zhang</td>
<td colspan="2">Rat - Y Dog - Y/N Human - Y</td>
<td colspan="3"><ul>
<li><p>ICAM-1 serves as a marker of endothelial cell activation (Cotran
RS (1987). New role for the endothelium in inflammation and immunity. Am
J Pathol 129:407-413)</p></li>
<li><p>ICAM-1 on the activated endothelial cells as a ligand interacts
with Mac-1 (CD11b/CD18), a leukocyte integrin receptor</p></li>
<li><p>ICAM-1 plays a role in margination and extravasation of
lymphocytes at sites of inflammation or a localized immune
response</p></li>
<li><p>Increased levels of soluble ICAM-1 in patients with vasculitis
(Tesar V, Jelinkova E, Masek Z, Jirsa M Jr, Zabka J, Bartunkova J,
Stejskalova A, Janatkova I, and Zima T (1998).</p></li>
<li><p>Up-regulated ICAM-1 expression on the endothelial cells of the
mesenteric artery in rat treated with SKF (Zhang et al)</p></li>
<li><p>Refs: Gonzalez-Amaro R and Sanchez-Madrid F (1999). Cell adhesion
molecules: selectins and integrins. Crit Rev Immunol. 19:389-429.
Coll-Vinent B, Grau JM, Lopez-Soto A, Oristrell J, Font C, Bosch X,
Mirapeix E, Urbano-Marquez A, and Cid MC (1997). Circulating soluble
adhesion molecules in patients with classical polyarteritis nodosa. Br J
Rheumatol. 36:1178-1183.</p></li>
</ul></td>
<td></td>
<td>B</td>
</tr>
<tr class="odd">
<td></td>
<td>VCAM-1</td>
<td>Zhang</td>
<td colspan="2">Rat - Y Dog - Y/N Human - Y</td>
<td colspan="3"><ul>
<li><p>A marker of endothelial cell activation</p></li>
<li><p>VCAM-1 on the activated endothelial cells as a ligand interacts
with VLA-4 (CD49d/CD29), leukocyte 41 integrin</p></li>
<li><p>VCAM-1 plays a role in the firm attachment and subsequent
transendothelial migration of leukocytes</p></li>
<li><p>Increased levels of soluble VCAM-1 in patients with vasculitis
(Ara J, Mirapeix E, Arrizabalaga P, Rodriguez R, Ascaso C, Abellana R,
Font J, and Darnell A (2001). Circulating soluble adhesion molecules in
ANCA-associated vasculitis. Nephrol Dial Transplant 16:276-285)</p></li>
<li><p>Refs: Ara J, Mirapeix E, Arrizabalaga P, Rodriguez R, Ascaso C,
Abellana R, Font J, and Darnell A (2001). Circulating soluble adhesion
molecules in ANCA-associated vasculitis. Nephrol Dial Transplant.
16:276-285. Gearing AJH and Newman W (1993). Circulating adhesion
molecules in disease. Immunol Today. 14:506-512. Gonzalez-Amaro R and
Sanchez-Madrid F (1999). Cell adhesion molecules: selectins and
integrins. Crit Rev Immunol. 19:389-429.</p></li>
</ul></td>
<td></td>
<td>B</td>
</tr>
<tr class="even">
<td><strong>Immediate Hypersensitivity -Pseudoallergen
(Burchiel)</strong></td>
<td>histamine</td>
<td>Burchiel</td>
<td colspan="2">Rat - NR Dog - Y Human - Y</td>
<td colspan="3"><ul>
<li><p>histamine is the main vasoactive mediator released from mast
cells and basophils in dogs and humans</p></li>
</ul></td>
<td></td>
<td>B</td>
</tr>
<tr class="odd">
<td>As discussed above for mast cell and basophil targets, current
research indicated that psudeoallergy may be a mechanism of altered flow
and permeability of vessels, as well as inflammatory mediator release;
there are numerous products of activated mast cells and basophils that
could be potential biomarkers; however, at this time none of these
markers have been associated with vascular injury</td>
<td>serotonin</td>
<td>Burchiel</td>
<td colspan="2">Rat -Y Dog - N Human - N</td>
<td colspan="3"><ul>
<li><p>serotonin (5HT) is the main vasoactive mediator released from
mast cells and basophils in rats and other rodents</p></li>
</ul></td>
<td></td>
<td>B</td>
</tr>
<tr class="even">
<td></td>
<td>PG's and LT's</td>
<td>Burchiel</td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td colspan="3"><ul>
<li><p>potential marker, but no data to date</p></li>
</ul></td>
<td></td>
<td>B</td>
</tr>
<tr class="odd">
<td></td>
<td>chymase</td>
<td>Burchiel</td>
<td colspan="2">Rat - Dog - Human -</td>
<td colspan="3"><ul>
<li><p>potential marker, but no data to date</p></li>
</ul></td>
<td></td>
<td>B</td>
</tr>
<tr class="even">
<td></td>
<td>tryptase</td>
<td>Burchiel</td>
<td colspan="2">Rat - Dog - Human -</td>
<td colspan="3"><ul>
<li><p>potential marker, but no data to date</p></li>
</ul></td>
<td></td>
<td>B</td>
</tr>
<tr class="odd">
<td><strong>Apoptosis (Nagarkatti)</strong></td>
<td>FasL</td>
<td>Nagarkatti</td>
<td colspan="2">Rat - Y Dog - Y/N Human - Y</td>
<td colspan="3"><ul>
<li><p>FasL is a 40-kd type 2 transmembrane protein belonging to the
tumor necrosis factor (TNF) family</p></li>
<li><p>ligation of Fas, a cell-surface protein transduces an apoptotic
signal leading to cell death</p></li>
<li><p>FasL is expressed mainly on activated leukocytes but recent
studies suggest that it may also be expressed on endothelial cells;
membrane bound FasL is rapidly cleaved into a soluble form which can be
detected in the serum (1)</p></li>
<li><p>Fas ligand has been implicated in endothelial cell injury (2,3).
Currently there are no tests in the market to detect soluble FasL in the
serum</p></li>
<li><p>Nagarkatti lab has published a paper on detection of mouse sFasL
using ELISA (1), and are currently developing an ELISA for rat FasL.
Zeytun A, Nagarkatti M, Nagarkatti PS</p></li>
<li><p>Growth of FasL-bearing tumor cells in syngeneic murine host
induces apoptosis and toxicity in Fas(+) organs. Blood. 2000 ; 95
:2111-7. Janin A, Deschaumes C, Daneshpouy M, Estaquier J,
Micic-Polianski J, Rajagopalan-Levasseur P, Akarid K, Mounier N,
Gluckman E, Socie G, Ameisen JC. CD95 engagement induces disseminated
endothelial cell apoptosis in vivo: immunopathologic implications.
Blood. 2002; 99:2940-7. Rafi AQ, Zeytun A, Bradley MJ, Sponenberg DP,
Grayson RL, Nagarkatti M, Nagarkatti PS. Evidence for the involvement of
Fas ligand and perforin in the induction of vascular leak syndrome. J
Immunol. 1998 ;161:3077.</p></li>
</ul></td>
<td>B</td>
<td></td>
</tr>
<tr class="even">
<td>Apoptosis may be a mechanism of target cell injury which may be
detected through various protein-based and biochemical assays,
cell-based flow cytometry, and immunohistochemical studies; it is also
possible that peripheral blood leuokocytes may serve as surrogates for
tissues undergoing apoptosis [Burchiel]</td>
<td>caspases</td>
<td>Nagarkatti</td>
<td colspan="2">Rat - Y Dog - Y/N Human - Y</td>
<td colspan="3"><ul>
<li><p>there are a series of caspases that are activated during
apoptosis that can be detected using cells undergoing apoptosis</p></li>
<li><p>this assay would require having sufficient numbers of recovered
apoptotic cells to perform the biochemical assays</p></li>
</ul></td>
<td>B</td>
<td></td>
</tr>
<tr class="odd">
<td></td>
<td>Annexin V</td>
<td>Burchiel</td>
<td colspan="2">Rat - Y Dog - Y/N Human - Y</td>
<td colspan="3"><ul>
<li><p>Annexin V is a useful marker for human and rodent
apoptosis</p></li>
<li><p>Annexin V binds to cell membranes that bleb during the apoptotic
process exposing phosphatidyl serine residues</p></li>
<li><p>Annexin V techniques have not proven useful in dog
studies</p></li>
</ul></td>
<td>B</td>
<td></td>
</tr>
<tr class="even">
<td></td>
<td>TUNEL</td>
<td>Burchiel</td>
<td colspan="2">Rat - Y Dog - Y/N Human - Y</td>
<td colspan="3"><ul>
<li><p>the TUNEL assay can be performed using both flow cytometry and
immunohistochemistry</p></li>
<li><p>it relies upon DNA repair enzymes that will incorporate
biotinylated UTP into DNA</p></li>
</ul></td>
<td>B</td>
<td></td>
</tr>
<tr class="odd">
<td><p><strong>Immune Mediated</strong></p>
<p><strong>(Miller and Zhang)</strong></p>
<p>Drug-induced vascular injury in man was initially described over
three decades ago and now over 50 chemical and biologic therapeutics and
vaccines have been associated with these syndromes. These agents can
induce a variety of vasculitic manifestations ranging from small vessel
hypersensitivity vasculitis and leukocytoclastic vasculitis to distinct
vasculitic syndromes such as Wegener's granulomatosis, polyarteritis
nodosa, and Churg Strauss syndrome. The agents associated with vascular
injury in humans do not share any obvious structural, pharmacological or
immunological properties. The pathogenic mechanisms remain to be defined
and appear to be multifactorial. Indirect evidence, however, implicates
chronic cellular and humeral immune activation in genetically
susceptible individuals. Some cases associated with urticaria,
angioedema, anaphylaxis and anaphylactoid reactions may involve Type I
immunoglobulin (Ig)-mediated or Type III hypersensitivity, or may be
caused by pharmacological, non-allergic means. Many drug-induced
vasculitis cases though are possible manifestations of immune complex
disease. These syndromes can be life-threatening diseases, and while
most cases respond to dechallenge (removal of the suspect agent), many
require additional concomitant immunosuppressive therapy for full
resolution. References: Cuellar ML. Drug-induced vasculitis. Curr
Rheumatol Rep 2002 Feb;4(1):55-9; D'Cruz D. Autoimmune diseases
associated with drugs, chemicals and environmental factors. Toxicol Lett
2000 Mar 15;112-113:421-32; Jain KK. Drug-induced cutaneous vascu-litis.
Adverse Drug React Toxicol Rev 1993 Winter;12(4):263-76; Dubost JJ,
Souteyrand P, Sauvezie B. Drug-induced vasculitides. Baillieres Clin
Rheumatol 1991 Apr;5(1):119-38</p></td>
<td>Autoantibody (ANCA); Immune complex &amp; anaphylatoxin</td>
<td><p>Miller</p>
<p>Zhang</p></td>
<td colspan="2">Rat - Y Dog - Y/N Human - Y</td>
<td colspan="3"><p><u>Myeloperoxidase (MPO) &amp; Proteinase 3
(PR3)</u></p>
<ul>
<li><p>Autoantibodies directed against cytoplasmic antigens of
neutrophils (ANCA), especially those with specificity for MPO and PR3,
are valuable markers for vasculitides.</p></li>
<li><p>ANCA are specific granule proteins of granulocytes and
monocytes</p></li>
<li><p>The cytoplasmic (classic) cANCA is induced by antibodies directed
against protainase 3 (PR3; PR-ANCA) while the perinuclear pANCA is
induced by antibodies against myeloperoxidase (MPO; MPO-ANCA) (Gross WL,
Schmitt WH, Crernok E (1993). ANCA and associated diseases:
immunodiagnostic and pathogenetic aspects. Clin Exp Immunol 91:1-12)
(Mayet WJ, Schwarting A, and Meyer zum Buschenfelde KH (1994). Cytotoxic
effects of antibodies to protainase 3 (C-ANCA) on human endothelial
cells. Clin Exp Immunol 97:458-465)</p></li>
<li><p>A positive correlation between MPO-ANCA and PR3-ANCA levels and
increased sE-selectin, sP-selectin, sICAM-1, and sVCAM-1 was reported in
patients with ANCA-associated vasculitis using the quantitative
anti-MPO-ELISA and anti-PR3-ELISA (Ara J, Mirapeix E, Arrizabalaga P,
Rodriquez R, Ascaso C, Abellana R, Font J, and Darnell A (2001).
Circulating soluble adhesion molecules in ANCA-associated vasculitis.
Nephrol Dial Transplant 16:276-285)</p></li>
<li><p>Cytokines (TNF-, IL-, increase PR3 expression in the
cytoplasm of endothelial cells. A possible direct pathogenic effect of
anti-PR3 antibodies, through ANCA-endothelial interaction, has been
suggested in vasculitides (Mayet WJ, Crernok E, Szymkowiak C, and Gross
WL, Meyer zum BUSCHENFELDE KH (1993). Human endothelial cells express
proteinase 3, a target antigen of anticytoplasmic antibodies in
Wegener's granulomatosis. Blood 82:1221-1229)</p></li>
<li><p>Direct cytotoxic effects of antibodies to PR3 on human vascular
endothelial cells a PR3 antibody-mediated mechanisms of endothelial
injury (Mayet WJ, Schwarting A, and Meyer zum Buschenfelde KH (1994).
Cytotoxic effects of antibodies to protainase 3 (C-ANCA) on human
endothelial cells. Clin Exp Immunol 97:458-465); Refs: Vorcheimer and
Fuster: JAMA 2001, 286:2154; Lindmark, et al: JAMA 286:2107; Zhang et
al: JAMA 286:2136</p></li>
</ul></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td><strong>Blood Coagulation Factors (Zhang)</strong></td>
<td>thrombomodulin</td>
<td>Schwartz/Zhang</td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td colspan="3"><ul>
<li><p>Important regulator of activated thrombin - TM converts thrombin
from a procoagulant to an anticoagulant</p></li>
<li><p>is a constitutive membrane protein - therefore probably needs to
be cleaved into soluble forms to be detected in circulation</p></li>
<li><p>increased circulating concentrations of an antigenic component
has been reported with thrompocytopenic purpura, disseminated
intravascular coagulation, atherosclerosis and inflammatory connective
tissues diseases</p></li>
<li><p>in vitro studies suggest TM is in endothelial cell tissue culture
supernatants suggesting comes from damaged endothelial cells; Other cell
sources - synovial cells, rheumatoid patients, platelets,
megakaryocytes, mesothelial cells, PMN's; unlike vWF, TM concentrations
appear independent of inflammatory cytokines IL-1 and TNF.; unknown if
assay works in humans- utility in rats [Schwartz]Boffa MC and
Karmochkine M (1998).</p></li>
<li><p>Refs: Thrombomodulin: An overview and potential implications in
vascular disorders. Lupus. Suppl 2: S120-S125. Boehme MWJ, Raeth U,
Scherbaum WA, Galle PR, and Stremmel W (2000). Interaction of
endothelial cells and neutrophils in vitro: Kinetics of thrombomodulin,
intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular
cell adhesion molecule-1 (VCAM-1): Implication for the relevance as
serological disease activity markers in vasculitides. Clin Exp Immunol.
119:250-254.</p></li>
</ul></td>
<td></td>
<td>B</td>
</tr>
<tr class="odd">
<td></td>
<td>vWF</td>
<td>Schwartz/Zhang</td>
<td colspan="2">Rat - Y Dog - Y/N Human - Y</td>
<td colspan="3"><ul>
<li><p>The activated endothelial cells release vWF into the plasma and
subendothelial matrix through exocytosis of the cytoplasmic
Weibel-Palade bodies</p></li>
<li><p>Exocytosis of vWF from Weibel-Palade bodies is associated with
the mobilization of P-selectin from Weibel-Palade bodies to the surface
of endothelial cells</p></li>
<li><p>High levels of vWF in patients with vasculitis</p></li>
<li><p>In endotoxin-treated or balloon-injured rats, the immunostaining
content of intracellular vWF was increased to three-fold, indicating
that endothelial cell injury leads to increased vWF levels (Reidy MA,
Chopek M, Cao S, McDonald T, and Schwartz SM (1989). Injury induces
increase of von Willebrand factor in rat endothelial cells. Am J Pathol
134:857-864)</p></li>
<li><p>Up-regulated vWF expression on the endothelial cells of the
mesenteric artery in rats treated with SKF (Zhang et al)</p></li>
<li><p>High molecular weight procoagulant product of endothelium - is a
constituent of platelets (alpha granules) and mRNA of vWF is found in
platelets, vWF can also be obtained from isolated platelets in
vitro</p></li>
<li><p>backers suggest that circulating vWF is specific marker of
endothelial damage and contribution from platelets is minimal. Platelet
vWF after discharge remains bound to platelet surface</p></li>
<li><p>component of Wiebel-Palady bodies in endothelial cells (storage
intracellular) also secreted constitutively secretion agonists -
thrombin, IL-1, vasopressin</p></li>
<li><p>Clinical evidence of increased circulating vWF - inflammatory and
atherosclerotic vascular diseases, after surgical procedures - bypass
surgery, infarction with ischemic heart disease, thromboembolic disease,
rheumatoid arthritis, increased with acute phase reactants; sensitive
marker - perhaps too sensitive 18 hr half life [Schwartz].</p></li>
<li><p>Refs: Newsholme SJ, Thudium DT, Gossett KA, Watson ES, and
Schwartz LW (2000). Evaluation of plasma von Willebrand factor as a
biomarker for acute arterial damage in rats. Toxicol Pathol. 28:688-693.
Cid MC, Monteagudo J, Oristrell J, Vilaseca J, Pallares L, Cervera R,
Font C, Font J, Ingelmo M, and Urbano-Marquez A (1996). von Willebrand
factor in the outcome of temporal arteritis. Ann Rheum Dis.
55:927-930.</p></li>
</ul></td>
<td>B</td>
<td></td>
</tr>
<tr class="even">
<td></td>
<td>Tissue Factor</td>
<td>Zhang</td>
<td colspan="2">Rat - Dog - Human -</td>
<td colspan="3"><ul>
<li><p>A procoagulant, not expressed in normal endothelial
cells</p></li>
<li><p>Cytokine-activated endothelial cells increase TF procoagulant
activity (Cotran RS (1987). New role for the endothelium in inflammation
and immunity. Am J Pathol 129:407-413)</p></li>
<li><p>A marker of endothelial cell activation</p></li>
<li><p>Increased levels of soluble TF in patients with vasculitis (Jurd
KM, Stephens CJM, Black MM, and Hunt BJ (1996). Endothelial cell
activation in cutaneous vasculitis. Clin Exp Dermatol 21:28-32)</p></li>
<li><p>Refs: Bach FH, Robson SC, Ferran C, Winkler H, Millan MT,
Stuhlmeier KM, Vanhove B, Blakely ML, van der Werf WJ, Hoffr E, de
Martin R, and Hancock WW (1994). Endothelial cell activation and
thromboregulation during xenograft rejection. Immunol Rev. 141:5-30.
information about time course of TF, TM, vWF in vitro study)</p></li>
</ul></td>
<td></td>
<td>B</td>
</tr>
<tr class="odd">
<td></td>
<td>tPA</td>
<td>Zhang</td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td colspan="3"><ul>
<li><p>An anticoagulant</p></li>
<li><p>Cytokine-activated endothelial cells decrease tPA anticoagulant
activity and increase plasminogen activating inhibitor-1 (PAI-1)
procoagulant activity</p></li>
<li><p>Increased levels of soluble tPA in patients with vasculitis (Jurd
KM, Stephens CJM, Black MM, and Hunt BJ (1996). Endothelial cell
activation in cutaneous vasculitis. Clin Exp Dermatol 21:28-32)</p></li>
<li><p>Refs: Cockwell P, Tse WY, and Savage COS (1997). Activation of
endothelial cells in thrombosis and vasculitis. Scand J Rhematol.
26:145-150. Jurd KM, Stephens CJM, Black MM, and Hunt BJ (1996).
Endothelial cell activation in cutaneous vasculitis. Clin Exp Dermatol.
21:28-32</p></li>
</ul></td>
<td></td>
<td>B</td>
</tr>
<tr class="even">
<td><strong>Hemodynamic (Schwartz)</strong></td>
<td>vascular bed pressure - flow or imaging</td>
<td>Schwartz-Burchiel</td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td colspan="3"><ul>
<li><p>vascular flow and pressure changes may or may not be predictive
of vascular injury – the techniques needed to measure flow in animals
are invasive and as such are not true biomarkers</p></li>
<li><p>in humans there are new imaging techniques that may be useful to
measure flow changes in regional vasculatures</p></li>
</ul></td>
<td>A</td>
<td></td>
</tr>
<tr class="odd">
<td><strong>New Technologies</strong></td>
<td>Proteomics</td>
<td>Sistare-Burchiel</td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td colspan="3"><ul>
<li><p>techniques such as Power Blotting, 2D gel electrophoresis and 2D
HPLC combined with SELDI, LC/MS/MS and MALDI techniques may be useful to
define profiles of proteins that may be associated with vascular
injury;</p></li>
<li><p>this work is more hypothesis-generating than hypothesis-driven
and is at an early stage of development</p></li>
<li><p>the hope is that novel protein markers may be discovered through
global scanning or proteins</p></li>
</ul></td>
<td></td>
<td>A</td>
</tr>
<tr class="even">
<td></td>
<td>Genomics</td>
<td>Sistare-Burchiel</td>
<td colspan="2">Rat - Y Dog - Y/N Human - Y</td>
<td colspan="3"><ul>
<li><p>techniques such as gene array (Affymetrix and other arrays) may
be useful to define profiles of genes that may be associated with
vascular injury</p></li>
<li><p>this work is more hypothesis-generating than hypothesis-driven
and is at an early stage of development</p></li>
<li><p>the hope is that novel gene markers may be discovered through
global scanning and that quantitative PCR technique can be developed to
monitor gene biomarkers</p></li>
</ul></td>
<td></td>
<td>A</td>
</tr>
<tr class="odd">
<td><p>Initial studies suggest that metabonomics NMR profiling and
computertechniques may be useful to non-invasively identify animals with
vascularinjury; however, the observed changes in biofluids may be
secondary or</p>
<p>tertiary to the mechanism of vascular injury or may be related to</p>
<p>physiological changes or non-vascular toxicity confounding
interpretation of</p>
<p>the data.</p></td>
<td>Metabonomics</td>
<td>Robertson</td>
<td colspan="2">Rat - Y Dog - Y Human - Y</td>
<td colspan="3"><ul>
<li><p>initial studies suggest that metabonomics NMR profiling and
computer techniques may be useful to identify animals with vascular
injury; however,</p></li>
<li><p>these techniques rely on nonspecific changes in kidneys and other
organs that may occur far downstream from the toxicologic events and
often do not allow for mechanistic interpretation</p></li>
</ul></td>
<td></td>
<td>A</td>
</tr>
</tbody>
</table>

# APPLICATION OF NEW TECHNOLOGIES TO UNDERSTAND PATHOGENSIS

## Toxicogenomics

### Background

Toxicogenomics is an emerging discipline that uses genomic tools
including gene expression profiling technologies to address problems of
toxicological significance. Several recent publications on
toxicogenomics have been published and can be reviewed for a more
detailed discussion of its general principles (16, 76, 96). As applied
to vascular injury, toxicogenomics can contribute to a mechanistic
understanding of the lesion, molecular basis of species differences, aid
in classifying various types of vasculopathy, development of gene-based
screens to improve selection of compounds for drug development, and
identification of more sensitive and specific biomarkers. To date no
reports specifically describing the use of toxicogenomics techniques for
vascular injury in preclinical animal models have been published.
However, there have been numerous publications from the field of
cardiovascular disease demonstrating that arterial tissue can be
characterized using expression profiling approaches, and therefore the
mechanistic understanding of vascular injury should be a realistic goal
(40, 102).

There are at least two ways that toxicogenomic approaches can facilitate
the identification of vascular injury biomarkers. First, by monitoring
genes that are differentially expressed in blood vessels isolated from
animals treated with compounds that induce vascular injury, it might be
possible to narrow the search for candidate biomarkers by specifically
focusing on genes that encode cell-surface or secreted proteins. These
genes encode potential circulating biomarkers that could be further
characterized directly in plasma or serum using immunoassays or other
diagnostic methods. Second, surrogate biomarkers might also be
identified by profiling circulating leukocytes, which can be easily
obtained from whole blood. Alcorta *et al.* (5) have reported that gene
expression changes in circulating leukocytes from patients with a
variety of renal diseases, including small vessel vasculitis (ANCA
disease), can be clustered according to disease type. The latter
approach will be particularly useful for characterizing vascular injury
in humans.

### Platform(s)

There are numerous technologies and platforms that can be used for
genome-wide expression profiling. While there are several ways to
categorize genomic approaches, most relevant for the study of vascular
injury are so-called "closed" and "open" profiling systems. Closed
systems typically use a microarray-type format and are based on
pre-defined sets of genes. Such genes could be selected to address a
specific hypothesis (e.g. oxidative-stress array), to characterize
certain tissues (e.g. kidney array), diseases (e.g. breast cancer
array), or profile an entire genome (e.g. rat genome array). Microarrays
are generally based on cDNA clones, PCR-amplified cDNA fragments, or
chemically synthesized oligonucleotides that are "spotted" or affixed to
a solid support such as a glass slide or a nylon membrane. These arrays
can be obtained from a variety of commercial vendors or constructed in
house using commercially available or homemade equipment. Another type
of array---the GeneChip from Affymetrix---is based on oligonucleotides
that are synthesized directly on a glass surface. Regardless of the
platform used, experiments typically involve isolating RNA from control
and treated tissues or cell lines, labeling the RNA, and hybridizing it
to the array. Depending on the specific platform, absolute or
differential expression levels of the genes represented on the arrays
are then calculated and used for statistical analysis. Because
mechanisms of vasculitis are not known, it is prefered to use the
genome-wide microarrays that are generally available for rat, mouse, and
human. Although these closed systems are evolving as the genomes become
better defined, genes critical to the pathogenesis of vascular injury
could be overlooked.

In contrast to the "closed" systems, "open" expression-profiling systems
do not begin with a pre-selected set of genes. Instead, the differences
between two populations of RNA are determined directly, generally using
some combination of RT-PCR, gel electrophoresis, and DNA sequencing.
Examples include SAGE (Serial Analysis of Gene Expression), differential
display, and subtractive hybridization. Kits for many of these
techniques are commercially available, and several biotechnology
companies use proprietary versions to build expression databases. One
advantage of open methods for characterizing vascular injury is that,
depending on the platform's sensitivity, most of the genes directly
involved in the lesion should be identified using these techniques; this
would include some genes that have not been previously identified and
therefore would not be represented on microarrays. Another advantage is
that they may be better suited for characterizing vascular injury in
species whose genomes have not been extensively sequenced, such as the
dog. Limitations of the open methods include their technical difficulty,
time-consuming nature, and expense.

### Knowledge Gaps/Limitations

There are a number of technical issues that must be considered when
working with blood vessels. Certainly one of the most significant is
that most blood vessels are small, making it difficult to obtain
sufficient quantities of RNA for most profiling methods. This problem is
of course more acute in rodents than dogs. The development of
increasingly sensitive amplification techniques offers promise that
starting RNA amounts will not be a limitation in the future. Moreover,
like most tissues, blood vessels are complex tissues comprised of
multiple cell types and can be found in close association with
surrounding connective tissues such as fat, pancreas or lymph nodes.
When interpreting expression data from vascular tissue, this complexity
can make it difficult to distinguish the contributions of endothelial
cells from vascular smooth muscle cells (VSMCs), infiltrating
leukocytes, and surrounding tissues. One possibility is to perform *in
situ* hybridization or immunohistochemistry to localize the cellular
source of specific transcripts of interest, assuming the necessary
antibody and cDNA reagents are available or can be generated. Another is
to use laser capture microdissection (LCM) to isolate "pure" populations
of endothelial cells, VSMCs, or other targeted cell populations (91).
Inherent to the lack of mechanistic understanding of vascular injury,
good in vitro models are not available, limiting the ability to generate
higher-throughput gene-based in vitro screens for vascular
injury-inducing potential.

As with all large-scale expression profiling experiments, data analysis
and interpretation remains a key challenge. Lists of genes that are up-
and down-regulated in tissues from animals with vascular injury will
easily be generated, but how can we separate cause from effect? This
task will be facilitated by solid experimental designs that include time
courses (i.e. take samples at early time points to capture potential
initiating events), dose-response (i.e. include a low dose that does not
cause toxicity to help separate pharmacologically mediated changes in
gene expression from toxicological ones), and careful choice of positive
and negative controls (i.e. generating expression data from animals
treated with compounds that cause inflammation but not vascular injury
will facilitate the search for more specific biomarkers). Furthermore,
bioinformatic approaches to link differentially expressed genes to
altered metabolic and signaling pathways and well-designed and focused
follow-up studies are critical to confirm new hypothesis that global
gene expression approaches might generate.

Ultimately, the success of identifying biomarkers for vascular injury
and understanding mechanisms of vascular injury may require application
of multiple technologies, including genomics, proteomics, metabonomics,
flow cytometry, imaging, etc. This creates the additional challenge of
combining disparate data from these various approaches and integrating
them to allow cross-platform querying and extraction of biological
knowledge to gain a more holistic understanding of vascular injury.

## Proteomics

### Background

Proteomics technologies are broadly characterized as tools designed to
examine the expression of proteins in a given set of samples. Types of
information derived from proteomics technologies include protein
identity, quantity, interactions, structure, and post-translational
modification. The nature of the proteome presents a series of challenges
that limit the ability of any single technology to completely assay and
characterize it. First and foremost is the heterogeneity of proteins.
Proteins can be large or small, globular or compact, hydrophobic or
hydrophilic, basic or acidic. Post-translational modifications such as
glycosylation, lipidation, or phosphorylation can have dramatic effects
on how the proteins behave in biochemical assays. A second challenge of
the proteome is the extraordinary wide dynamic range of protein
expression, which is estimated to be one billion fold (21). This
presents an extraordinary challenge to any detection method. A third
challenge of the proteome is the absence of a method of direct
amplification. While the polymerase chain reaction can amplify DNA and
RNA, no such technique exists for proteins (although there are promising
approaches in development). These challenges together create a large
opportunity for technology development, as well as niches for the myriad
of technologies that have already been developed. For the researcher
studying vascular injury, these technologies can provide powerful tools
to discover biomarkers or to understand fundamental mechanisms. To date
there have been no published reports detailing proteomic--derived
biomarkers for vascular injury, however a number of laboratories are
currently pursuing the approach.

### Platforms (Strengths/Weaknesses)

This document cannot, of course, list all proteomic technologies, which
are constantly being developed and refined. Instead, it will focus on
several well-established techniques that are broadly aimed at expression
profiling (although some have additional applications such as studying
post-translational modification). Consequently, a discussion of
high-throughput crystallography, as an example, will not be presented
here. Several themes will develop. First, all proteomics technologies
consist of some permutation of separation followed by detection. Second,
no single proteomics technology in its current state can possibly hope
to characterize the entire proteome. It is therefore the recommendation
that each researcher apply multiple approaches to the study of the
proteome.

Classically, two-dimensional gel electrophoresis (2-D PAGE) has been
used to assay the proteome (1). Protein samples are first separated on
the basis of pI using an isoelectric focusing gradient. These proteins
are then further separated in a second dimension based on their
migration in an electric field, which is generally a function of their
hydrodynamic volume, which roughly correlates to their molecular weight.
Detection can then be performed using any of a myriad of agents,
including silver staining and fluorescent dyes. Individual spots on the
gels can be excised, digested with trypsin, and tryptic fragment
fingerprint matched against a database. Where indicated, tandem mass
spectrometry can be performed and matched against the theoretical tandem
mass spectra of peptide sequences to provide (in most situations)
definitive identification. Its drawbacks include poor visualization of
low abundance proteins, generally poor visualization of extremely basic
or acidic proteins, poor visualization of hydrophobic proteins, and low
throughput (36). Matching of 2D gels from different samples is
time-consuming, although computer algorithms that aid this process are
improving. Thus, this technique is useful for the researcher who wishes
to perform differential profiling and has abundant sample volume but
limited numbers of samples. It is not useful for the researcher with
limited sample volume or large numbers of samples. This may limit the
statistical validity of some of the conclusions derived from 2D gel
studies. Recent advances in software used to align 2D gels, integrated
platforms, and independent 2D gel service providers can overcome many of
these limitations.

Surface-enhanced laser desorption ionization and time-of-flight mass
spectrometry (SELDI-TOF MS) is a proteomics technique with a broad range
of applications, including protein expression profiling (29). Separation
is performed using a combination of traditional elution chromatography
(usually on spin columns) followed by retentate chromatography.
Retentate chromatography is performed by incubating samples (or
fractions of samples) on arrays containing chromatographic substrate on
its surface (ref). Traditional chromatographic steps of binding and
washing are performed, but what is detected by laser desorption TOF MS
are the proteins that have been retained on the arrays. This technique
can be used for differential profiling, particularly when samples are
scarce (only 20 ul of serum is required) as well as when a relatively
large number of samples need to be profiled. In addition to differential
profiling, this technology can be used to study protein-protein
interactions, post-translational modifications, and epitope mapping.
Therefore, it may be quite useful in studying mechanisms of vascular
injury in addition to identifying novel biomarkers. Its principle
drawback is that biomarker identification of the biomarkers generally
requires some form of off-line purification, although identification
itself is possible using the array.

A relatively novel technique, ICAT ™ for differential expression
profiling requiring labeling of samples has recently been described and
commercialized. In this method, the proteins in two samples to be
compared are labeled separately on the side chains of their reduced
cysteinyl residues using one of two isotopically different, but
chemically identical sulfhydryl-reactive ICAT reagents (one being an
isotopically "light" reagent, d(0), the other being a "heavy" reagent
containing eight deuterium atoms on its carbon backbone, d(8)). The
labeled protein mixtures are combined and enzymatically digested (e.g.
with trypsin), and the labeled peptides are isolated by affinity
chromatography, using the affinity tag (biotin group) that is part of
the ICAT reagents. The selected peptides are separated by liquid
chromatography and analyzed by tandem mass spectrometry (37). This
technique assumes quantitative and equivalent labeling and recovery of
analytes and labels only proteins containing cysteines. In addition, it
is not well suited to the study of proteins that have been modified
post-translationally. It may be a good technique for differential
profiling of a limited number of samples, but is not high-throughput.

Multidimensional liquid chromatography followed by tandem mass
spectrometry is another technique for identification proteins contained
in a complex sample (34). Samples for this procedure are generally
globally digested, and then liquid chromatography using orthogonal
separations principles is performed serially, and the individual
peptides are then sequenced by tandem mass spectrometry. While useful in
providing information of proteins contained within a sample,
quantitation information is minimal and the procedure is notoriously
low-throughput.

Finally, a brief discussion of protein arrays in their many forms is
warranted (98). There has been much interest in developing arrays on
which hundreds to thousands of proteins or antibodies are immobilized,
and then incubated with a sample and binding information for each of
these analytes obtained simultaneously. This has, however, been
difficult to achieve in practice, in large part because identifying
reagents (i.e. purification of the thousands of proteins or antibodies)
is difficult, immobilization strategies that retain protein function are
difficult to achieve, and various protein-protein interaction reactions
often have differing binding requirements (e.g. pH, salt, divalent
cations, etc). Specific types of protein arrays include tissue arrays
and multi-analyte western blots (refs). In the future, it can be
expected that many types of arrays will be developed, most with specific
types of applications rather than global protein expression profiling.

These descriptions are not intended to be comprehensive, but rather
insight into the major proteomic tools available to the researcher
studying vascular injury. The vascular injury researcher should be aware
of these diverse technology offerings. Some of these techniques are
readily transferable to the individual researcher's laboratory; others
are best done in collaboration with experts versed in the use of the
techniques. In addition, the vascular injury researcher will find many
opportunities to participate in the improvement of existing techniques
and the development of novel ones. Beyond the applications of protein
expression profiling detailed here, the study of protein-protein
interactions to delineate protein-interaction maps can be quite useful
in studying the mechanisms underlying vascular injury. In addition, the
study of post-translational modifications is a core application of many
of these proteomics technologies and can therefore be useful in studying
how different types of post-translational modification determine the
function of the proteins involved in the mechanisms of vascular injury.

## Metabonomics 

### Background and Principles 

Toxicants, by definition, disrupt the normal composition and flux of
endogenous biochemicals in, or through, key intermediary cellular
metabolic pathways. These disruptions alter, either directly or
indirectly, the blood that percolates through the target tissues. This
altered blood can then produce urine changes, either directly or
indirectly, producing characteristic biomolecular traces. The diagnostic
utility of any one trace biomolecule is limited due to the number of
variables affecting it along its route to the urine and by the
commonality of biochemical processes disrupted by toxicants. However, if
a significant number of trace molecules are monitored, the overall
pattern, or " fingerprint" produced may be more consistent and
predictive than any one marker. Metabonomics combines the techniques of
high-resolution nuclear magnetic resonance (NMR) and pattern recognition
technology to rapidly generate these diagnostic metabolic fingerprints
(39, 71, 72, 82).

### Platforms

Although, in principle, many analytical techniques could be used for
metabonomic evaluations, high field NMR provides the distinct advantage
of being able to simultaneously detect thousands of molecules in a
bodily fluid with little sample preparation. This ability, allows an
unbiased assessment of metabolic response (54, 55). Proton (^1^H) NMR
spectroscopy is capable of detecting individual atoms in soluble
proton-containing molecules with a molecular weight of approximately 20
kD or less. Standard flow probes used with 600 MHz spectrometer are
capable of detecting thousands of resonances in the urine from molecules
at concentrations in the mid micromolar range or higher with a data
acquisition time of a few minutes. Incorporation of cryoprobe technology
will lower this limit significantly. The other half of the technology,
pattern recognition, is considerably more diverse in the availability of
platforms (27, 41). However, principal component analysis (PCA) has been
the most frequently used technique for small scale focused metabonomic
studies (84). An exciting area of ongoing research is the linking of
metabonomics technology with proteomic and toxicogenomic platforms
allowing potential biomarker assessment from gene to protein to
phenotype.

### Application of Technology to Drug-Induced Vasculopathy

The application of metabonomic technology to drug-induced vasculopathy
is a relatively recent development. To date most work has focused on
PDE4- induced arteriopathy in the rat. Principal component analysis
produced a clear urine spectral pattern separation between 8 of 11 rats
with vascular lesions and 36 of 37 rats without lesions in samples
collected after 3 or 4 days of treatment with CI-1018, a PDE4 inhibitor
(83). Furthermore, in rats immunosuppressed with dexamethasone, the
urine spectral pattern was still evident, suggesting the spectral
changes were not simply an indirect index of inflammation (89). The
specificity of the spectral changes produced in rats with vascular
lesions was confirmed by Zhang et al (103), who demonstrated that
spectral changes produced in response to renal lesions in SHR rats could
be differentiated from spectral changes associated with vascular lesions
produced by the same compound in WKY rats. Work with rolipram,
demonstrated that the spectral changes precede vascular lesions in both
time and dose (82).

### Advantages/Disadvantages 

Using urine as a sample matrix, a distinct advantage of the technology
is that comprehensive toxicological information can be gained from a
single animal including onset, peak and regression of toxicity without
concerns over sample numbers or timing. Results can be readily
correlated with concurrent clinical and clinical pathology assessments
if necessary. Since sample collection is non-invasive, analyses are
easily piggybacked onto other studies with minimal impact. Sample size
is usually not an issue in rats as they produce 10 --20 mL of urine/day.
Sample preparation is minimal requiring only addition of antibacterial
agent, dilution in deuterated buffer and centrifugation to eliminate
particulates - all of which are amenable to robotic processing in a
microtiter plate format. The analyses themselves can be conducted
relatively rapidly (200-300/day) and have a relatively low sample
cost/analysis. Since NMR is non-selective, once a sample is analyzed,
the spectra can be queried as needed to obtain different biological
information without the necessity of rerunning the analyses. The primary
disadvantages of the technology are the high initial capital costs of
obtaining an instrument and the necessity for collecting samples in
metabolism cages with cold collection capability as bacterial
contamination of samples essentially eliminates the utility of the
sample. Other disadvantages with the technology are not appreciably
different than those associated with proteomic and genomic approaches.
Since NMR is non-selective, any change in animal physiology or toxicity
(other than the one of interest) may affect the urine NMR spectra,
making it difficult to associate a pattern specifically with the lesion
of interest. Additionally, it is often difficult to mechanistically
associate bimolecular changes in urine to observed lesions. NMR is not
particularly sensitive (relative to something like mass spec) but it is
not subject to interferences and the utilization of longer acquisition
times or the use of cryoprobe technology can reduce the sensitivity
limitation. Given the wealth of data that can be generated and the
uncertainty as to what it all means, bioinformatics limitations and
information overload plague this technology as they do the other "omics"
approaches.

### Knowledge Gaps: 

Metabonomics is a newer technology than proteomic and toxicogenomic
approaches (at least to the toxicologist anyway). With that newness
comes a certain amount of "black box" fear for those not familiar with
NMR. However NMR, as a platform, is much older and more stable than
platforms for other technologies, which should help reduce those fears
accordingly. The multivariate statistical methods are much less user
friendly and represent a real knowledge gap between the users and those
assessing the significance of the generated data. Within the vascular
injury application, the primary knowledge gap is the lack of a clear
mechanistic relevance of the biomolecular changes driving the pattern
separation. It seems clear that the urinary changes are not simply a
reflection of inflammation, but it is difficult to understand how
minimal to moderate pathologic changes in one vascular bed (e.g. the
mesentery) could drive micro to millimolar urinary changes in molecules
involved in intermediate metabolism. It seems likely that other factors
may be involved, some of which, when elucidated, may lead us to a better
understanding of the etiology of this troublesome lesion in rats which
may lead to a clearer evaluation of the human clinical significance of
these changes.

## Flow Cytometry 

### Background and Principles

There are numerous circulating cells present in the blood stream of both
clinical and pre-clinical species. These cells are exposed to the
pharmacologic agent of interest, and the possibility exists that during
vascular toxicity, cells within the vessel wall may dislodge and enter
the circulation. Information, both quantitative and functional, can be
obtained by analyzing these circulating or dislodged cells.. There is
considerable heterogeneity between arterial, venous, and capillary
vascular endothelium, and even organ specific endothelial phenotypic
alterations have been identified (19). This may allow discrimination of
site-specific vascular injury, once appropriate markers have been
identified. Flow cytometry has wide application in the study of
circulating cells, which can be collected by minimally invasive means.
It permits rapid simultaneous acquisition of multiple parameters, which
are valuable for analyzing diverse functional or quantitative changes in
individual cells (86). With the advent of eight- or nine-color sorters
and/or analyzers permitting multicolor fluorescence analyses and the
continuing development and commercialization of a wide range of
fluorescent probes have made flow cytometry a unique technology to meet
these needs. These capabilities coupled with sequential gating
strategies permit analyses of extremely large numbers of cells leading
to subsequent identification of even rare cells (32).

### Platforms

Flow cytometry allows for rapid measurement of numerous characteristics
on individual cells/particles moving single file in a fluid stream.
Forward angle light scatter can distinguish cell/particle size whereas
side angle light scatter can determine internal complexity. Furthermore,
light emitted from fluorescently labeled probes or antibodies allows
identification of a wide variety of cell surface, cytoplasmic and
nuclear structures. Light scatter and fluorescent emission are then
detected by photodetectors. Flow cytometers use two basic platforms,
bench top analyzers and sorters. These are available from a variety of
manufacturers. Both platforms can be used for high speed laser cell
interrogation and analysis The biggest difference is that a sorter can
be used to select cells of interest into a receptacle for further
analysis or culture. Typically sorters are much more accommodating in
design of lasers and optics compared to bench top analyzers, providing
more flexibility in study design, and numbers of parameters that can be
examined simultaneously.

### Application of Technology to Drug-Induced Vasculopathy

The application of flow cytometry to the study of drug induced vascular
injury has only begun recently. Flow cytometric analysis in relation to
drug-induced vascular injury would be applicable in several areas 1)
alterations of normally circulating cells such as white blood cells, 2)
cells released from the vascular wall into circulation after vascular
damage, 3) assessment of oxidative stress in circulating cells. Single
cell suspensions of circulating leukocytes can be monitored as sentinels
of drug-induced effects as it lends itself amenable to rapid analyses of
thousands of cells (32). There are numerous commercially available
antibodies to cell surface proteins on leukocytes in most species,
allowing for immunophenotyping and assessment of activation markers. It
is currently unknown how drug-induced vascular damage impacts these
measurements. There are small numbers of circulating endothelial cells
present in normal individuals, and the numbers have been shown to
increase after various insults resulting in vascular damage (23). To
date, most of these studies have utilized antibody linked magnetic beads
to concentrate circulating endothelial cells for enumeration, and the
use of flow cytometric quantitation is untested. There is *in vivo*
evidence for oxidative stress in vascular endothelium and leukocytes of
spontaneously hypertensive rats as compared to normotensive rats (94).
Hypertension is well known to result in vascular damage and repair. This
assay relies on the use of hydroethidine that is a reduced
non-fluorescent precursor of the fluorescent molecule ethidium bromide.
Hydroethidine, in the presence of superoxide radical, converts to
ethidium bromide that can bind DNA and then fluoresces brightly. This
assay could be applied to models of drug-induced vascular damage to
assess either leukocytes or circulating endothelial cells.

### Advantages/Disadvantages

Advantages. The equipment is widely available in both clinical and
preclinical laboratory settings. There is an ever-increasing number of
commercially available antibodies and fluorochromes applicable to flow
cytometry. Additionally, antibodies can be made to most antigens
providing increased utility to flow cytometric analyses in toxicology.
Flow cytometric examination of cells can be done at rates of tens of
thousands of cells per second, and can frequently be done in an
automated fashion.

Disadvantages. The capital costs for equipment, especially for sorters,
are quite high. Qualified personnel are needed to run and interpret
multi-parameter data. Antibodies and fluorochromes are key to fully
utilizing the potential of flow cytometry in assessment of vascular
damage. Despite the availability of antibodies for endothelial cells,
because of the heterogeneity of endothelial cells, truly pan-endothelial
antigens likely do not exist. This could also be an advantage once the
localization of antibodies is mapped to specific vascular endothelium,
potentially allowing identification of vascular damage location. Making
new antibodies to current antigens without antibodies or previously
unidentified antigens takes both time and money. Many of the currently
available antibodies do not cross-react across multiple species.

### Knowledge Gaps

It is currently unknown whether localized vascular damage, typical of
the drug-induced vasculopathies, alters either normally circulating
cells or dislodges vascular wall cellular components in a detectable
fashion. Likewise the duration of increased or altered circulating cells
after vascular insult is largely unknown, although it appears to return
to normal as the symptoms subside (33, 70, 88, 90). There is marked
disagreement, in the literature, on the number of circulating
endothelial cells present in normal individuals; these discrepancies may
be related to differences in methods and/or antibodies utilized. It will
be important to provide details regarding methodology for quantitation
of cells when reporting on models of vascular damage. There is currently
a need to more fully elucidate the phenotypic heterogeneity of the
vascular endothelium in a localized manner, specifically in vascular
beds where drug-induced vascular damage is common: coronary vascular bed
in dogs, splanchnic vascular bed in rats. Localization of damage is
currently only detected by histologic examination. Reagents allowing
this localization should be pursued by techniques such as injection of
phage-display peptide libraries that detect specific surface molecules
in the vascular endothelium (74), or pursuing novel methods of culturing
endothelial cells from specific vasculature (25, 62).

## Bioinformatics and Multivariate Statistics

### Background

"Omics" dominates the list of new techniques available to the researcher
studying almost any biological system. Rapid developments in technology
platforms coupled with high throughput techniques allow researchers to
measure many analytes from many samples simultaneously, generating a
staggering amount of data. This leads to the challenge of mining that
data, archiving that data, and retrieving that data. The fields of
bioinformatics and multivariate statistics, coupled with increasing
computational power, present to the researcher solutions to their
data-mining problems as well as opportunities for research and
development of novel solutions to these problems. Because of this,
computational biology should itself be thought of as a novel technology.

### Platforms

The term bioinformatics in this discussion is reserved strictly for two
tasks: first, to describe methods that mine primary and secondary
sequences of nucleic acids and proteins as well as structural data for
proteins to derive information regarding genes or proteins; and second,
to archive and retrieve such data in a computationally efficient manner.
Thus, bioinformatics encompasses strategies used to perform sequence
alignment, to predict coding regions from genomic DNA, predict secondary
structure of proteins, and so forth. It also encompasses databasing such
information, such as is performed at the National Center for
Biotechnology Information (NCBI) or SWISS-PROT. Another type of database
is one of theoretical enzymatic digests of proteins contained in a
protein database, which is searched with an actual enzymatic digest of
an unknown sample to obtain the identity of the protein. An outstanding
review text is written by Baxevanis (8). Vascular injury researchers
have at their disposal a number of tools, many of which are continually
being improved. For example, better prediction of coding regions in
genomic data leads to a more complete catalog of genes. This in turn
leads to a more complete database of enzymatic digests for protein
identification. A more complete understanding of protein function and
structure allows researchers to catalog protein domains and sites of
specific types of post-translational modification, thus allowing a
researcher to generate and test hypotheses regarding protein function
more quickly. However, much remains to be done. For example, databases
of theoretical translations do not account for real or theoretical
post-translational modifications. This can lead to an incorrect protein
identification or, more likely, the mistaken conclusion that one has
found a heretofore unidentified protein. Algorithms that more accurately
predict gene structure also remain to be developed. Bioinformatics can
also be used to understand species differences in vascular injury (i.e.
comparative genomics). For example, sequence alignment can reveal
conserved domains that might dictate similar functions (and therefore
similar responses to drugs) as well as non-conserved regions that might
help predict divergent functions.

This discussion will not include the problem of visualization of
genomics or proteomics data. A good review can be found in Chakravarti
(17). Perhaps of more interest to the researcher employing these "omics"
technologies is statistical analysis of data generated by these
techniques. For example, if one were to use a 10k cDNA microarray on 20
samples, there would be 200,000 data points. SELDI technology can easily
generate data for 140,000 protein peaks for forty samples. Particularly
in the most common employment of these "omics" technologies, there is a
profound asymmetry between sample number (few) versus data points per
sample (many). This presents a challenge for statisticians, in which the
converse situation (many data points relative to sample number) is
preferred. Generally, the goal is to find the set of variables (genes or
proteins) that distinguish classes (such as with vascular injury versus
without vascular injury) of samples from each other. Univariate analysis
on a variable-by-variable basis is simple to perform, but in most
complex biological conditions, it is unusual to find a single variable
that is both sensitive and specific for a given condition. For example,
a number of acute phase reactant proteins are increased in animal models
of vascular injury, but these are generally not considered to be
specific to vascular injury (?reference?). Using multiple variables
simultaneously is a common method to overcome the limitation of single
markers. Multivariable analysis is therefore an important component of
any study aimed at identifying biomarkers of vascular injury.
Multivariate analysis can be conveniently divided into two broad
classes, supervised and unsupervised. Supervised learning requires that
during the development of a classification model, information regarding
outcome (class assignment) is available and is useful in developing a
diagnostic. Unsupervised learning does not have such a requirement and
is more powerful for data exploration. These classes of algorithms will
be discussed in turn.

Supervised learning techniques are generally described as classification
algorithms, in which data for each of the samples is supplied to the
algorithm along with the class assignment for each sample (classes are
the various groupings of samples such as drug-treated versus untreated).
The algorithm uses the class assignment to develop models that can be
used to classify these samples (called the training set). In the ideal
case, these models are then tested on different samples for which the
identity is hidden (called the testing set). In general, the performance
of the model on the test set is expected to be lower than that on the
training set. The quality of the model is a function of the overall
classification success rate as well as the decline in classification
success rate from the training set to the test set of samples. It is not
uncommon to develop a model that can classify the training set with high
success but that does poorly on a test set -- these models are generally
described as being overfit. Examples of supervised learning techniques
include support vector machines, neural nets, and decision trees. The
vascular injury researcher is recommended to utilize algorithms that
provide the underlying variables that contribute to the classification
model, since these features can be concluded to be biomarkers for future
workup.

Unsupervised learning techniques are generally described as clustering
algorithms. No class assignments are presented to the algorithm, which
attempts to distribute the samples as far apart as possible in
n-dimensional space. Samples that are more similar to each other will be
closer to each other in these models, and the distance between samples
is therefore a measure of dissimilarity. Common unsupervised learning
techniques include self-organizing maps, hierarchal and k-means
clustering, and principal component analysis. These types of techniques
are particularly useful in finding hidden classes. Since no a priori
assumptions are made about the categorization of samples, the
distribution of the samples may reveal heretofore-unknown subgroups. A
drawback of unsupervised learning techniques is that deconvolution of
these models may not reveal a defined, small subset of biomarkers.
Instead, one often gets a long list of co-regulated genes or proteins.
This can be particularly problematic for discovery of biomarkers that
are to be used as a framework for mechanistic studies of vascular
injury.

Statisticians are constantly developing refinements of these techniques
as well as novel approaches. The challenge for the vascular injury
researcher not versed in statistics will be to utilize the technique(s)
that are most appropriate for the type of data being mined. Vascular
injury researchers should seek out collaboration with statisticians who
are versed in the biology of genes and proteins, so that intelligent
decisions regarding algorithm selection and parameters are made.
Moreover, it should be recognized that proper experimental design is the
first step toward optimal statistical analysis; poor experimental design
can lead to the inability to make statistically sound conclusions or,
worse, incorrect conclusions from the data. Finally, all data that are
supplied to these types of algorithms must be intelligently
pre-processed (e.g. calibrated and normalized)(28).

### Conclusions

The technologies described in this section can be sources of biomarkers
as well as provide insight into the mechanisms of vascular injury. The
combination of these technologies will provide a synergistic effect,
both from the standpoint of validating results across the various
platforms, as well as providing the opportunity for "meta" statistical
analysis i.e. the integration of information garnered from multiple
techniques. It is recognized that the adoption of any one of these
technologies is itself a resource heavy proposition, and to undertake
more than one simultaneously creates even greater pressure on
potentially limited resources. Therefore, it would be prudent for
researchers across institutions to allocate resources and become centers
of excellence in a specific subset of these technologies. The data can
then be shared, archived, and mined. This will expedite the discovery
and validation of biomarkers and enable a greater understanding of the
mechanisms of vascular injury.

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