Optical molecular imaging of lysyl oxidase activity - detection of active fibrogenesis in human lung tissue

Article abstract of DOI:10.1039/c5sc01258a

Aberrant fibrogenesis is a feature of many diseases in multiple organ systems. The lysyl oxidase family of enzymes are central to tissue homeostasis and elevated lysyl oxidase activity is implicated in fibroproliferation as well as in cancer stroma. We have synthesised a novel fluorogenic reporter for monitoring lysyl oxidase activity that generates a 3-5 fold increase in fluorescence following probe activation in ventilating fibrotic ex vivo asinine lung and ex vivo human lung tissue. The probe termed "oLOX" can provide real-time measurement of lysyl oxidase activity in a number of biological settings and is tractable from an in vitro setting to man.

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Optical molecular imaging of lysyl oxidase activity –
detection of active fibrogenesis in human lung tissue
a#
b#
a#
Tashfeen Aslam, Amy Miele, Sunay V. Chankeshwara, Alicia Megia-
a
b
b
b
b
Fernandez, Chesney Michels, Ahsan R Akram, Neil McDonald, Nik Hirani,
Chris Haslett, Mark Bradley, Kevin Dhaliwal.
b
a*
b*
Aberrant fibrogenesis is a feature of many diseases in multiple organ systems. The lysyl
oxidase family of enzymes are central to tissue homeostasis and elevated lysyl oxidase
activity is implicated in fibroproliferation as well as in cancer stroma. We have synthesised
a novel fluorogenic reporter for monitoring lysyl oxidase activity that generates a 3-5 fold
increase in fluorescence following probe activation in ventilating fibrotic ex vivo asinine
lung and ex vivo human lung tissue. The probe termed “oLOX” can provide real-time
measurement of lysyl oxidase activity in a number of biological settings and is tractable
from an in vitro setting to man.
Introduction
same enzymatically active C-terminus and the cofactor lysine
1
0,11
tyrosylquinone.
A by-product of all of these enzymes is
Aberrant fibrogenesis is a feature of many diseases in multiple hydrogen peroxide. Lysyl oxidases are important in cancer
organ systems and is characterised by tissue injury, remodelling progression and in many fibrotic diseases including those
1
2–15
and incomplete repair resulting in the excessive deposition of affecting the lung.
Of the 5 members of the LOXF, two in
1
,2
collagen. Fibrogenesis is a prominent feature of several lung particular have been the basis of extensive investigation and
diseases ranging from adult respiratory distress syndrome in therapeutic targeting: LOX and LOXL2. LOX expression
intensive care to chronic obstructive pulmonary disease and correlates with poor survival and is a therapeutic target for
interstitial lung diseases. The global impact of these diseases is patients with certain cancers, while LOXL2 is a focus in the
huge, causing significant financial burden. Despite this, monitoring and treatment of fibrotic lung disease.
3
14
4
15,16
treatment options are very limited and monitoring of disease As the potential for lysyl oxidases as therapeutic targets
progression remains a challenge, as there are no rapid bedside becomes clear, there is a discernible need for the development
biomarkers that can detect active fibroproliferation. Blood of selective and sensitive oxidase substrates to monitor
biomarkers offer poor molecular specificity for lung pathology, enzymatic activity in complex biological systems. As a result,
while pulmonary biopsy is invasive and fixed tissue cannot fluorescence-based analysis methods have attracted much
5
inform on the dynamic enzyme activity that exists in vivo.
attention as they can provide simple, effective and powerful
1
7
The Lysyl oxidases are copper dependent amine oxidases tools for real-time monitoring of LOX enzyme activities in
6
18–20
that play a central role in fibrogenesis. They facilitate the vitro and in vivo.
Until recently, such reporters have been
covalent cross-linking found within elastin and collagen by designed either on a coumarin21,22 or resorufin scaffold and
catalysing the oxidative deamination of peptidyl-lysine and are based on a single amine oxidation/β-elimination resulting in
hydoxylysine residues, a step that is crucial to the mature and an amplification of fluorescent signal. However, the excitation
23
7
–9
functional extracellular matrix. There are five proteins within wavelength for coumarin (λex 360nm) limits its applicability
the lysyl oxidase family (LOXF): LOX, LOXL1 (Lysyl oxidase for cellular-based imaging. Amplex red (N-acetyl-3,7-
like- one), LOXL2, LOXL3 and LOXL4, all of which share the
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Optical molecular imaging of lysyl oxidase activity
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dihydroxyphenoxazine) is a non-fluorescent derivative of alkylation of fluorescein gives the dual ether/ester (
dihydroresorufin that is converted to fluorescent resorufin on major product, rather than the bis-ether ( ). To develop the
This reagent has been optimal conditions to synthesise the desired bis-ether product
utilised in a number of settings including the quantification of reactions conditions were optimised looking various solvents
6) as the
5
2
4,25
reaction with hydrogen peroxidase.
1,
4
1
neutrophil NADPH oxidase, monamine oxidase (MAO), and bases. It is known that the use of heterogeneous Ag(I)
1
9,24,25
glucose oxidase and LOX.
While providing a sensitive salts favours the desired alkylation reaction over the
2
6
41
42
method for the detection of hydrogen peroxide , Amplex red is acylation, and the use of silver oxide in acetonitrile with
non-specific and clearly cannot be used in vivo or in cells.
1
9
molecular sieves was effective for the synthesis of 5 in
In a recent publication, Li et al reported a probe for the moderate yield (Scheme 1b). The aprotic solvent maintaining
fluorometric detection of monoamine oxidases A and B in vitro. fluorescein in its closed form, with the crude reaction mixture
The probe was synthesised over 5 steps using the familiar and affording
inexpensive dye, fluorescein . While utilising the amine non-fluorescent while quinoid
oxidation/β-elimination mechanism in the presence of MAO-A characteristics (λmax = 520nm) of fluoresein. Treatment of
and B, the fluorescent reporter probe was methyl fluorescein, a with hydrochloric acid in DCM/ether gave reporter as its HCl
In order to develop a salt in quantitative yield. As anticipated, the final probe had
probe for demonstrating the activity of LOXF in vivo, a reporter good aqueous solubility and was non-fluorescent.
probe with higher quantum yield was required. Herein, we The two specific and potent LOX inhibitors 10 and 11 were
5
and
6
in 15% and 65% yields, respectively.
5 was
3
6
showed emission
5
1
2
7
dye with a lower quantum yield than 4.
report the synthesis and ex vivo biological evaluation of an synthesised. 11 was designed based on structure activity
easily synthesised activity-based fluorescent probe for LOXF relationship of similar inhibitors where the introduction of 4-(p-
that utilises a fluorescein scaffold.
To validate the activation of the optical LOXF probe (oLOX)
fluorophenyl)phenol significantly enhanced the potency of the
inhibitor . 2-phenyl-3(2H)-pyridazinones, were synthesised in
3
2
1
in the presence of LOX, selective inhibition was desired. β- two steps by condensation of mucochloric acid with aryl
Aminopropionitrile (BAPN) has been extensively used as the hydrazine to afford the dichloro pyrazone derivative . In the
next step, nucleophilic substitution with N-Boc piperazine
Though BAPN is widely used as a specific irreversible inhibitor derivative afforded . Reaction of with 4-phenylphenol or 4-
indeed its (p-fluorophenyl)phenol and Boc deprotection afforded the
7
2
8
reference LOX inhibitor, with a reported IC50 of 10 µM.
8
8
2
9,30
of LOX, it does have affinity for other oxidases,
structure suggests non-specificity. Over the last two decades inhibitors 10 and 11 in good yields (50-55%).
selective inhibitors of LOX, as anti-fibrotic agents, have been
3
1, 32, 33, 34, 35
developed.
are among the most potent (IC 0.005-0.07µM) . Compounds
Of these the pyridazinone-based class LOX and LOXL2 are expressed in human lung tissue
3
6
5
0
LOX is important in lung development and is expressed in
3
7
1
0
(IC50 3nM) and 11 were thus synthesized for application in
inhibition studies.
oLOX was evaluated for LOX activity in freshly isolated
human lung tissue, showing for the first time, the ability to
measure the dynamic activity of LOXF with optical detection in
this model and the potential for in vivo utility.
43,44
normal lung tissue.
Pathological activity of LOXL2 has
been shown by Barry-Hamilton et al with increased expression
of LOXL2 in the lung tissue of patients with idiopathic
15
pulmonary fibrosis (IPF), while Chien et al found an
association between serum LOXL2 levels and the progression
16
of IPF. Increased expression of LOX has also been well
demonstrated in experimental models of pulmonary
Results and discussion
45,46
fibrosis.
Hence we wished in particular to begin evaluating
our probe in the setting of human lung pathology.
Synthesis of optical LOXF probes
The expression of LOX and LOXL2 was firstly confirmed
The chemical “masking” of the phenol groups of fluorescein in human lung tissue homogenates using western blot and
3
8,39
can suppress fluorescence and facilitate cell permeability.
this investigation, both phenolic groups of were selectively cells then cleaved to the 32kDa mature protein by bone
alkylated with aminopropyl moieties, it being hypothesised that morphogenetic protein-1. In this study we demonstrated the
upon treatment of oLOX with the appropriate oxidases, the presence of the 50kDa pro-LOX protein and the active 32kDa
two amino groups would be oxidised to give the bis-aldehyde
LOX protein in human lung tissue as well as the enzymatically
followed by dual β-elimination to release acrolein and active 63kDa LOXL2 (Figure 1). Immunohistochemical data
fluorescein (Scheme 1a).
supported previous findings whereby LOX and LOXL2 were
The synthesis of oLOX
of xanthene dyes such as fluorescein, which exists in either of were expressed in human lung tissue, homogenised lung tissue
the two forms, the quinoid and/or lactone form, depending was deemed an appropriate model to evaluate the utility of the
on the pH and solvent environment. The fluorescent, xanthen- activity-based sensor oLOX
-one tautomer form, is favoured in non-acidic aqueous
solutions; whereas a non-fluorescent, lactone tautomer , is
favoured at acidic pH’s or in non-aqueous solvents. Typically
In immunohistochemistry. The 50kDa pro-LOX is secreted from
3
1
0
1
2
4
1
5
1
was complicated due to the nature found to be expressed in lung tissue. Since LOX and LOXL2
4
3
4
0
1
.
3
3
2
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a) Activation of probe oLOX
1 by LOX
b) Activity-based probe
1
Scheme 1 a: Pathway for the fluorescence activation of probe oLOX
1
by LOX.
b
: Synthetic route for oLOX, i) Br(CH ) NHBoc,
2
3
Ag O, MeCN, py, 4Å MS, 40°C, 48 h, (15%) and 6 (65%); ii) HCl, DCM/ether, 2 h, 93%.
5
2
LOX inhibitors 10 (Inh-1) and 11 (Inh-2)
Scheme 2 Synthetic route to the LOX inhibitors. i) Mucochloric acid, 50% aq. MeOH, 3h, 60%; ii) N-Boc-piperazine, NaI,
dioxane, 100 °C, 20 h, 70%; iii) bisphenol derivative, CuI, Cs CO , BINOL, dioxane reflux, 12h, 58-73%; iv) 20% TFA in DCM,
2
3
1
h, quantitative.
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Optical molecular imaging of lysyl oxidase activity
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MAOB inhibitor) did not cause any significant reduction in
fluorescent signal (Figure 3). Furthermore, BAPN does not
5
4
inhibit MAO , thus confirming that MAO does not cleave
this model.
1 in
Fig. 3 oLOX is not activated by monoamine oxidases. Human
Fig. 1: LOX and LOXL2 are expressed in aged human lung
tissue homogenates. (a). Immunohistochemical analysis and (b).
western blot of LOX and LOXL2 expression in aged (55-81yr) human
lung tissue. Strongest bands for LOX were at 50kDa, consistent with
the glycosolated pro-lysyl oxidase, but mature 32kDa LOX was also
present in several samples (ESI, Figure S1). Data representative of a
lung homogenate was pre-incubated (in triplicate) with 500µM of
BAPN, clorgyline and/or pargyline at 37°C for 1 hour prior to the
addition of 10µM oLOX. The bar show the mean resultant fluorescence
intensity after 2 hour incubations (n=3). ***p<0.001, one way
ANOVA, dotted line represents background fluorescence of oLOX,
error bars depict standard error of the mean.
minimum of 3 experimental replicates. Scale-bar = 100m.
oLOXF detects increased LOXF activity in aged human tissue
and is inhibited by selective inhibitors
Incubation of oLOX with tissue homogenate resulted in up
to a 300% increase in fluorescence (Figure 2a). The temporal
kinetics of oLOX activation with human tissue at the
concentrations used in this study showed significant
fluorescence amplification after 30min (p<0.01) and plateaued
between 50min and 2h (ESI Figure S2). Importantly, oLOX
Optical Molecular Imaging of LOX
Whilst fluorometric determination of LOX in human tissue
has been demonstrated (Figure 2), the pivotal advance would be
the dynamic imaging of LOX activity in tissue in situ without
the need for homogenisation. This heralds the capability to
perform optical molecular ‘biopsies’ in situ with optical
activation was completely inhibited in
a concentration
dependent manner by pre-incubation of the homogenate with
BAPN (Figure 2b) and the inhibitors 10 and 11 (Figure 2c and
5
5,56
molecular imaging techniques
. Fibred confocal fluorescence
microscopy (FCFM) allows a flexible optical fibre bundle to be
passed down the working channel of an endoscope while
utilising laser excitation at 488nm, allowing visualisation of
lung elastin auto-fluorescence. FCFM has been successfully
utilised in a number of organ systems including the pulmonary
2
d). As predicted inhibitor 11 was a more potent inhibitor of
probe activation (Figure 2d). BAPN is a time and temperature
dependant irreversible inhibitor of LOX
shown to inhibit LOXL1 , LOXL3 and LOXL4 , but
inhibition of LOXL2 by BAPN is more controversial with
mixed reports of success.
4
7
48,49
. BAPN has been
5
0
51
52
5
7,58
2
6,53
tract
, where the autofluorescence of the elastin allows
We have shown that both active
visualisation of tissue architecture. This approach allows the
real-time in vivo visualisation of tissue at a cellular level and is
a clinically applicable strategy.
LOX and LOXL2 are present within the lung tissue
homogenates and hypothesise that each of these enzymes
contribute to the increase in fluorescent signal observed with
our probe. While we have not investigated the expression of
other members of the LOXF within the lung tissue, it is
certainly possible that they are present at low levels and
contribute to the fluorescent signal.
Hence we evaluated oLOX
1 in conjunction with FCFM, as an
optical molecular imaging strategy by applying oLOX to aged
human lung tissue ex vivo. To demonstrate that the resultant
increase in fluorescence detected by FCFM could be inhibited
and prove the specificity of oLOX, we also incubated human
lung tissue with BAPN (Figure 4).
While a probe of related structure has previously been used
2
7
to report on levels of MAO , the potential contribution of
MAO to oLOX activation in human lung tissue was negated by
the fact that clorgyline (a MAOA inhibitor) and pargyline (a
4
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Fig. 2 oLOX is activated in the presence of aged human lung tissue homogenate as shown by an inhibitable increase in
fluorescence. (a) Fluorescent intensity of 10µM oLOX after 2h incubation with aged human lung tissue homogenate (solid line). Spectra were
recorded with λex =450nm. (b-d) Human lung homogenate was pre-incubated with dilutions of inhibitors at 37°C for 1h prior to the addition of
1
0µM oLOX. Each graph shows the resultant fluorescence presented as percentage above background after 1h incubation of the reaction
(
representative of n=3). The minimum concentration of BAPN (b) and Inh-1 (c) found to have a significant inhibitory effect was 62µM (p<0.01,
one way ANOVA), whereas the minimum concentration of Inh-2 (d) found to have a significant inhibitory effect was 15 M (p<0.01, one way
ANOVA). The dotted line represents background fluorescence of oLOX, error bars depict standard deviation.
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Moving from sections of tissue (Figure 4) to whole organ and spontaneous model negated the need for an experimental
imaging was the next challenge. Hence, in order to assess the model under the 3Rs (Replacement, Reduction and Refinement
utility of oLOX in situ, a ventilating ex vivo asinine lung ethos. A significant increase in fluorescent signal over
model was used (Figure 5 and ESI Figures S3-4). We have background was detected following the installation of oLOX
)
1
1
recently reported that aged donkeys suffer from a high into fibrotic regions of whole ventilating ex vivo asinine lung
prevalence of pulmonary fibrosis (35%) (often resulting in that were subsequently confirmed to express LOX (Figure 5c).
euthanasia), which has been likened to a fibrotic interstitial lung The coupling of oLOX
disease of humans. A whole donkey lung was thus used to invasive visualisation of temporal and spatial alterations in the
1
with FCFM enables the minimally
5
9
assess the utility of combining FCFM with the local molecular activity of LOXF
intrapulmonary delivery of oLOX. Utilising this size relevant
.
Figure 4: oLOX can be used alongside fibred confocal fluorescence microscopy (FCFM) to demonstrate oLOX activity in
†
whole ex vivo human lung tissue. (a) Architecture of the FCFM (Cellvizio®-Lung System (Mauna Kea Technologies, Paris, France)
involving a fibre bundle and scanning confocal microscopy (b) The tip of the fibre optic is superimposed on a fixed lung tissue section with the
white bar corresponding to the diameter of the fibre (1.4mm). (c) Representative single frame images generated by FCFM on fresh sections of aged
human lung tissue following incubation with 10µM oLOX. The graph (d) represents the mean auto-fluorescence of each 60 second video imaging
period adjusted for tissue auto-fluorescence (n=4). Note the significant reduction in fluorescence on incubation with the lysyl oxidase inhibitor,
BAPN (*p = 0.0286, Wilcoxon signed rank test).
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Figure 5: oLOX can be used to image LOXF activity in a fibrotic ventilating ex vivo asinine lung model. Catheter installation of
00µM oLOX instilled in a total volume of 1 mL in PBS into a whole fibrotic ventilating ex vivo asinine lung results in a significant increase in
2
fluorescent signal (a-b, *p= 0.0313, Wilcoxon signed rank test, n=3); (c). Target tissue was evaluated for LOX expression using
immunohistochemistry (20X lens). (d) Western blot confirmed expression of LOX in both fibrotic and grossly unaffected (control) areas of ex vivo
asinine lung tissue.
enzymes is a future goal, exploiting the use of specific inhibitor
based imaging agents. A further goal is to
Conclusions
We have successfully designed and synthesized an activity- develop oLOX sensors that permit more rapid fluorescent
based fluorescent probe capable of the real-time quantification amplification.
of LOXF activity in fibrogenic conditions. The activation of the
probe by LOXF was confirmed in human lung homogenate
models. Furthermore, probe activation was inhibited in the
Acknowledgements
presence of a specific LOX inhibitor, thus confirming target
specificity. The probe also showed utility in a size-relevant
The authors would like to thank the MRC, under the
Developmental Pathway Funding Scheme (grant number
model of lung fibrogenesis. This optical Smartprobe has the
RA2159). A.M-F. acknowledges Fundacion Ramon Areces-
potential to image real-time LOXF activity within the lungs of
Postdoctoral Research Fellowship Program for financial
patients. Significant signal amplification is detected after
support. The research leading to these results has received
30min, a time-point that is feasible for bedside imaging.
.
funding from the European Union Seventh Framework
Programme FP7 2012 under grant agreement no. 326465 (A.
M-F.) and EPSRC (EP/K03197X).
Whilst we have not yet assessed the specificity of oLOX to
individual members of the LOXF family, targeting individual
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