Keywords
3F7 monoclonal antibody - abdominal aortic aneurysm - atherosclerosis - coagulation
factor XII - vulnerable plaques
Introduction
The rupture of an atherosclerotic plaque and formation of an occlusive arterial thrombus
are the inciting events leading to myocardial infarction and ischemic stroke, which
represent the leading causes of mortality and morbidity worldwide.[1] Atherosclerosis, the leading pathology that causes these ischemic complications,
is fuelled by chronic inflammatory processes.[2] While the role of the coagulation system initiating pathological thrombus formation
upon plaque rupture is well appreciated, there is now a growing body of evidence that
the coagulation system also directly contributes to atherosclerosis development, including
plaque destabilization.[3]
[4] Indeed, multiple coagulation factors have been demonstrated to be present within
atherosclerotic plaques.[5] Moreover, active coagulation factors such as tissue factor, thrombin, factor X (FX),
and factor XII (FXII) have been shown to be present early on during the development
of atherosclerotic plaques.[5] Supporting this notion, it has recently been demonstrated that genetic deficiency
of FXII or FXI inhibits atherogenesis in a mouse model of atherosclerosis.[6]
[7] These findings support the concept of utilizing anticoagulants as a means to prevent
atherosclerosis.[8] However, given that all clinically available anticoagulant therapeutics are associated
with an inherent risk of bleeding, newer more advanced strategies avoiding bleeding
are required.[8]
[9]
FXII, the zymogen of the serine protease, FXIIa, is activated by several substances
including polyphosphate, vascular collagen, misfolded proteins, and neutrophil extracellular
traps.[10]
[11]
[12]
[13] The activation of FXII initiates the intrinsic coagulation pathway and liberates
the formation of bradykinin (BK) thus promoting inflammation.[14] Significantly, recent data have demonstrated that FXII−/− mice are afforded protection from atherosclerosis.[6] Interestingly, the protective effects of FXII deficiency appear to occur independently
of any systemic effects on coagulation, or BK production, thus suggesting an important
proinflammatory role of FXII/FXIIa within the confines of the atherosclerotic plaque.
In this regard, FXIIa induces the production of proinflammatory, proatherogenic cytokines,
including interleukin (IL)-6, IL-1b, IL-12, and tumor necrosis factor-α from bone
marrow-derived macrophages.[6] However, to date, the effects of pharmacological FXIIa inhibition as an antiatherosclerotic
approach have not been investigated. FXIIa is a highly attractive therapeutic target
given the fully recombinant human 3F7 monoclonal antibody (mAb), which binds to the
catalytic site of activated FXII and potently inhibits both FXIIa and βFXIIa, and
has been demonstrated to inhibit thrombosis without impeding hemostasis in preclinical
animal models[15]
[16] and an affinity-improved version (CSL312) is currently in phase 3 clinical trials
for the prevention of hereditary angioedema (ClinicalTrials.gov Identifier: NCT04656418).
Therefore, given its key role in mediating pathological thrombosis and several inflammatory
processes implicated in the development of atherosclerosis and AAA, we hypothesized
that the therapeutic inhibition of FXIIa with the mAb 3F7 represents a novel approach
to inhibit atherosclerosis and AAA by treating both coagulation and inflammation,
while preserving hemostasis.
In this study, we demonstrate that inhibition of FXIIa with 3F7 has a protective effect
in mouse models of AAA and atherosclerosis. Most importantly, using a unique mouse
model of atherosclerotic plaque instability,[17]
[18]
[19] we show that FXIIa inhibition via 3F7 leads to the stabilization of vulnerable,
unstable atherosclerotic plaques. Taken together, these data indicate that FXIIa inhibition
suppresses thromboinflammation and thereby exerts beneficial effects in vascular diseases,
such as AAA and atherosclerosis.
Methods
Mouse Experiments and 3F7 Administration
All animal procedures were approved by the Animal Ethics Committee of the Alfred Medical
Research and Education Precinct (AMREP), Melbourne, Australia, under ethics application
numbers E/1658/2016/B and E/1187/2012/B and conform to the current National Institutes
of Health Guidelines for the Care and Use of Laboratory Animals. ApoE−/− mice were generated from a C57BL/6 background, bred, and maintained at the AMREP
Animal Centre.
Briefly, mouse models of AAA, stable, and unstable atherosclerosis were employed ([Supplementary Fig. S1], available in the online version). For the investigation of atherosclerotic plaques,
ApoE−/− and tandem stenosis (TS) mouse models were employed while an angiotensin II (AngII)-infusion
mouse model was used for the AAA study. Animals were randomly assigned to receive
either 3F7 or its isotype control BM4 every second day via intraperitoneal (i.p.)
injection, with administration beginning directly after surgery or at 8-weeks old
for the ApoE−/− study. Both 3F7 and BM4 (MuBM4-MuG1K) were supplied by CSL Limited as murine IgG1
antibodies.
Detailed information on the methods used in this publication can be found in the [Supplementary Material] (available in the online version).
Statistical Analysis
Unless otherwise specified, quantitative data are expressed as mean ± standard deviation.
Comparisons of parameters between two groups were made using the unpaired Student's
t-test after normal data distribution was confirmed. A p-value of <0.05 was considered statistically significant. All statistical analyses
were performed using GraphPad Prism software.
Results
FXIIa Is Localized within AAAs
Several negatively charged surfaces which can activate FXII have been described within
the AAA microenvironment.[20]
[21] Therefore, we postulated that FXIIa is detectable in AAAs. Using immunofluorescence
(IF), we confirmed the presence of FXIIa within AAAs ([Fig. 1A, B]).
Fig. 1 Presence of FXIIa in AAA and inhibition of FXIIa decrease severity and increase stability
of AAA. (A) Representative IF images showing FXIIa (3F7-staining) deposition within the AAA.
(B) BM4 was used as isotype control. The vessel lumen is marked by interrupted lines.
ApoE−/− mice were infused with angiotensin-II via an implanted osmotic minipump and then
treated with 3F7 or the control BM4. Ultrasound measurements of the dilatation of
the abdominal aorta were taken. (C) Percentages of ruptured, large, small, and no aneurysm formation (BM4 n = 11; 3F7 n = 12). (D) Ratio change (endpoint/baseline) for mice that developed AAA and survived, i.e.,
did not experience rupture (p = 0.0072; BM4 n = 7; 3F7 n = 10). (E) Table showing the incidence of IMT formation, determined by morphological assessment
or necropsy (BM4 n = 10; 3F7 n = 11). Representative images showing the morphological analysis of collagen deposition
using MTC and PSR for: (F) lower limit aneurysms from 3F7-treated animals (n = 5) and (G) a lower limit aneurysm from BM4 control animals (n = 1). The unpaired Student's t-test was used for comparison of BM4 and 3F7-treated mice. Scale bars = 200 µm. AAA,
abdominal aortic aneurysm; FXII, factor 12; FXIIa, activated factor 12; IMT, intraluminal
thrombosis; L, lumen; MTC, Masson's trichrome; PSR, Picro-Sirius Red.
Anti-FXIIa (3F7) Administration Results in Smaller AAAs and a More Stable Phenotype
We next investigated the efficacy of the anti-FXIIa mAb 3F7 to prevent the development
of AAA. Mice received 3F7 or BM4 isotype control antibody every 48 hours for 28 days
following the implantation of an osmotic minipump containing AngII to induce AAA.
Although 3F7 did not significantly alter the incidence of aneurysm formation, it did
result in less acute aortic ruptures and a higher proportion of small aneurysms on
the lower limit of classification (lower-limit aneurysm 1.2–1.4 mm, aneurysm >1.5 mm;
BM4 no aneurysm n = 1, lower-limit aneurysm n = 4, aneurysm n = 3, rupture n = 3; 3F7 no aneurysm n = 1, lower-limit aneurysm n = 9, aneurysm n = 1, rupture n = 1; [Fig. 1C]). Ultimately, inhibition of FXIIa significantly impeded the progressive dilatation
of the abdominal aorta throughout the 28-day experimental period, therefore forming
smaller aneurysms as compared with the control animals receiving BM4 ([Fig. 1D]; p = 0.0072). Individual plots of the baseline and endpoint dilatation measurements
can be found in [Supplementary Fig. S2A] and [B], available in the online version.
Studies have suggested that the volatility of an AAA and its risk of acute rupture
are correlated to the size and reactivity of the aneurysms' thrombus.[22]
[23] The AngII mouse model of AAA is characterized by the development of an intramural
thrombus (IMT) in the absence of an intraluminal thrombus.[24] Although FXIIa inhibition did not impede IMT formation ([Fig. 1E]), morphological analysis of those aneurysms containing an IMT demonstrated a more
stable phenotype in animals receiving 3F7 versus the control, BM4. Specifically, the
IMT of 3F7-treated animals with lower-limit small aneurysms showed thick deposition
of collagen between IMTs and the vessel–lumen interface ([Fig. 1F]), which was not seen in either lower-limit aneurysms ([Fig. 1G]) or aneurysms ([Supplementary Fig. S2C], available in the online version) from the BM4 control group. The formation of this
protective layer of collagen may offer a potential mechanism by which inhibiting FXIIa
might protect from AAA rupture.
Inhibition of FXIIa Promotes Stable Atherosclerosis
Following the observation of reduced dilatation and rupture, and evidence of increased
collagen content in AAA, we next explored the potential for 3F7 to promote stable
atherosclerosis. We investigated the effect of FXIIa inhibition on the development
of atherosclerosis in ApoE−/− mice fed a high-fat diet. We found that 3F7 decreased total atherosclerotic plaque
size and necrotic core area ([Fig. 2A, B]; p = 0.0031; 0.0489) and increased total collagen content ([Fig. 2C]; p = 0.0169), as compared with those mice receiving isotype control antibody BM4. No
significant changes in lipid profile were observed ([Supplementary Fig. S3], available in the online version). This demonstrates the protective effect of blocking
FXIIa, a finding consistent with a previous study demonstrating that genetic deficiency
of FXII in FXII−/−ApoE−/− double-knockout mice reduced the development of atherosclerosis.[6]
Fig. 2 FXIIa inhibition attenuated the development of stable atherosclerosis. ApoE−/− mice were placed on an HFD for 8 weeks to develop stable atherosclerosis. (A) H&E was used to morphologically assess plaque size (p = 0.0031), (B) necrotic core area (p = 0.0489), and (C) the collagen content (p = 0.0169) in stable plaques of the aortic sinus, as compared with the BM4 control–treated
animals (BM4 n = 6; 3F7 n = 5). Values are mean ± SD. Scale bars = 200 µm. Assays were assessed using unpaired
Student's t-tests. Interrupted lines indicate total lesion area used for collagen content analysis.
Representative images show one valve of the aortic sinus only. All valves were included
in the analysis. H&E, hematoxylin and eosin; HFD, high-fat diet; SD, standard deviation.
FXIIa Is Localized within the Microenvironment of Mouse-Unstable Atherosclerotic Plaque
To assess the efficacy of 3F7 in a more translationally relevant animal model of atherosclerosis,
we utilized a TS mouse model, which is characterized by thin-capped, rupture-prone,
unstable carotid artery plaques with a phenotype similar to the culprit lesions often
responsible for ischemic events in patients.[17] Anatomical information regarding the location of the TS and the predefined area
of plaque instability within this model can be found in the Methods section and Chen
et al.[17]
Prior to investigating the therapeutic capacity of 3F7 in the TS mouse model, we confirmed
the accumulation of FXIIa within the unstable atherosclerotic plaques. Fluorescence
(AF546)-labeled 3F7 was detected inside the unstable atherosclerotic plaques using
both IF ([Fig. 3A, B]) and in vivo imaging system ([Fig. 3C, D]). This is an important finding both in regard to demonstrating a pathological activation
of FXII in unstable plaque and also in regard to the suitability of FXIIa targeting
as a selective therapeutic approach.
Fig. 3 FXIIa accumulates abundantly in unstable atherosclerotic plaques as compared with
stable plaques. TS mice were injected with fluorescently labeled (AF546) 3F7 or BM4
and the presence of FXIIa was detected in unstable and stable atherosclerotic plaques
using IF microscopy and IVIS. Representative IF images show: (A) the presence of FXIIa detected by in vivo labeling within the unstable plaque of
a TS mouse; (B) a section of unstable plaque from an animal injected with BM4 isotype control. The
atherosclerotic vessels, both stable (segment V & TA) and unstable plaques (segment
I), as well as healthy vessels (segment IV), were excised and imaged using IVIS (left) and brightfield (right). (C) Segment I shows strong accumulation of fluorescently labeled 3F7 but not the isotype
control fluorescence antibody. Areas of stable atherosclerosis also reveal some FXIIa
binding, but to a lesser extent. FXIIa, activated factor 12; IVIS, in vivo imaging
system; TA, thoracic aorta; TS, tandem stenosis.
3F7 Stabilizes Vulnerable Atherosclerotic Plaques
Inhibition of FXIIa via administration of 3F7 began 24 hours after TS surgery. We
assessed various markers of plaque stability. At the endpoint of this study, there
were no differences in the body weight, spleen weight, or serum lipids between animals
receiving 3F7 and BM4 ([Supplementary Fig. S4], available in the online version). 3F7 administration decreased the total plaque
area, necrotic core size, and lipid deposition within the vulnerable plaques, as compared
with animals receiving BM4 ([Fig. 4A]–[C]; p = 0.0101; 0.0391; 0.0429). Furthermore, there was a marked increase in total collagen
deposition within the vulnerable plaques of those animals receiving 3F7, as well as
increases in cap thickness and cap-to-core ratio, two important measurements of plaque
instability directly translatable to clinical measurements of plaque instability in
humans ([Fig. 4D]–[F]; p = 0.0004; 0.0197; 0.0015). This increase in collagen deposition was mirrored by an
increase in intimal smooth-muscle cells, another structural element supporting atherosclerotic
plaque stability ([Fig. 4G]; p < 0.0001). Another important sign of plaque instability is intraplaque hemorrhage.
The antibody TER119 specifically binds to the glycophorin A-associated protein (Ly-76)
expressed on erythrocytes and so can be used to detect intraplaque hemorrhage. Mice
treated with 3F7 showed markedly reduced staining of TER119 in segment I of the TS
model as compared with those mice receiving the isotype control antibody BM4 ([Fig. 4H]; p = 0.0029), again strongly indicating the plaque-stabilizing effect of 3F7.
Fig. 4 Inhibition of FXIIa via antibody 3F7 stabilizes vulnerable atherosclerotic plaques.
ApoE−/− mice were placed on an HFD for 6 weeks prior to TS surgery. Following surgery, animals
remained on the HFD for 7 weeks and 3F7 or BM4 isotype control was administered. (A) H&E was used to morphologically assess plaque size (p = 0.0101; BM4 n = 16; 3F7 n = 17) and (B) necrotic core area (p = 0.0391; BM4 n = 14; 3F7 n = 19) in unstable plaques, as compared with the BM4-treated animals. (C) Oil-red O staining was used to assess the lipid deposition within the plaques (p = 0.0429; BM4 n = 15; 3F7 n = 18). Picro-Sirius Red was used to assess the degree of protective collagen deposition
within the plaques, with histological examination performed using BF and polarized
light. (D) The total intimal collagen deposition, (E) fibrous cap thickness, and (F) cap-to-core ratio, as compared with BM4-treated animals (p-values = 0.0004; 0.0197; 0.0015; BM4 n = 16; 3F7 n = 20). (G) Intimal smooth-muscle cell deposition (p = < 0.0001; BM4 n = 12; 3F7 n = 18) and (H) intra-plaque hemorrhage, as indicated by erythrocyte/TER119 staining (p = 0.0029; BM4 n = 12; 3F7 n = 11). Isotype refers to the isotype control for the antibody used in the respective
immunohistochemical stain. Assays were assessed using unpaired Student's t-tests. Values are mean ± SD. Scale bars = 200 µm. α-SM, α-smooth muscle; BF, brightfield;
FXII, factor 12; FXIIa, activated factor 12; H&E, hematoxylin and eosin; HFD, high-fat
diet; SD, standard deviation.
3F7 Administration Decreases Plaque Macrophage Content and Circulating Proinflammatory
Markers, as well as Increasing Markers Associated with Fibrosis in Vulnerable Plaques
Dual inhibition of prothrombotic and inflammatory pathways is a unique feature of
3F7. The effect of FXIIa inhibition via 3F7 on inflammation was assessed locally using
immunohistochemistry and systemically using a mouse cytokine/chemokine multiplex array.
Assessment of localized inflammation showed a marked decrease in VCAM-1 expression
following 3F7 treatment ([Fig. 5A]; p = 0.0002), which correlated to a reduction in macrophages infiltrating into the plaque
([Fig. 5B]; p ≤ 0.0001). Multiplexing identified significant downregulation of circulating chemoattractants:
monocyte chemoattractant protein-1 (MCP-1, CCL2), eotaxin-1 (CCL11), keratinocyte-derived
chemokine (CXCL1), and lipopolysaccharide-induced CXC chemokine (CXCL5), in the plasma
of animals treated with 3F7 ([Fig. 5C]; p = 0.0133; 0.0350; 0.0493; 0.0421). In addition, circulating proinflammatory markers
IL-1α, IL-1β, IL-12p40, and IL-17 ([Fig. 5D]; p = 0.0335; 0.0499; 0.0485; 0.0347), and stimulating factors granulocyte-macrophage
colony-stimulating factor and macrophage colony-stimulating factor were downregulated
([Fig. 5E]; p = 0.0459; 0.0234), while factors associated with wound healing and fibro-protection,
IL-4, IL-13, and IL-5, were all significantly upregulated following treatment with
3F7 ([Fig. 5F]; p = 0.0491; 0.0410; 0.0055). Interestingly, the level of circulating BK as determined
by liquid chromatography–mass spectrometry/mass spectrometry (LC-MS/MS; [Supplementary Fig. S5], available in the online version) was not influenced by 3F7 administration.
Fig. 5 Inhibition of FXIIa via 3F7 reduces plaque-localized and systemic inflammation. Immunohistochemistry
of unstable plaques shows: (A) reduction in VCAM-1 expression and (B) CD68+ macrophage infiltration by 3F7 administration (p = 0.002; <0.0001; BM4 n = 15–18; 3F7 n = 16). Isotype refers to the isotype control for the antibody used in the respective
immunohistochemical stain. In addition, further insights into the degree of inflammatory
markers circulating in the sera were obtained: (C) the chemoattractants MCP-1 (CCL2), eotaxin-1 (CCL11), KC (CXCL1), and LIX (CXCL5)
are significantly reduced (p-values = 0.0133; 0.0350; 0.0493; 0.0421), (D) the proinflammatory markers IL-1α, IL-1β, IL-12p40, and IL-17 are significantly
reduced (p-values = 0.0335; 0.0499; 0.0485; 0.0347), (E) the stimulating factors GM-CSF and M-CSF (p-values = 0.0459; 0.0234) and (F) the factors associated with wound healing and fibrosis, IL-4, IL-13, and IL-5 (p-values = 0.0491; 0.0410; 0.0055), are also significantly reduced (BM4 n = 12–24; 3F7 n = 17–24). Values are mean ± SD. Assays were assessed using unpaired Student's t-tests. Scale bars = 200 µm. FXIIa, activated factor 12; GM-CSF, granulocyte-macrophage
colony-stimulating factor; KC, keratinocyte-derived chemokine; LIX, LPS-induced CXC
chemokine; MCP-1, monocyte chemoattractant protein-1; M-CSF, macrophage colony-stimulating
factor; SD, standard deviation.
Discussion
Cardiovascular disease (CVD) remains a leading cause of mortality and morbidity globally.
With the rising diabetes and obesity epidemic in the developed world, in addition
to the significant risk of recurrent events for patients with CVD, there remains a
pressing need for novel drugs for CVD treatment and prevention. Moreover, therapeutic
options for patients with AAA remain limited to surgical options, which are often
inappropriate for a significant proportion of patients with AAA and are also associated
with high mortality rates. Therefore, given this unmet clinical need, nonsurgical
preventive and treatment options for AAA are required.[25] The availability of therapies that have the potential to inhibit the development
of atherosclerosis and to stabilize unstable atherosclerotic plaques, as well as rupture-prone
AAAs, would represent a major clinical need. Atherosclerosis and AAA are both characterized
by chronic inflammation, with single-cell RNA-sequencing studies beginning to highlight
the significant diversity of immune cells in both diseases.[26]
[27]
[28] Similarly, thrombosis is also a central feature of both pathologies.[29]
[30] As a result, therapies targeting both inflammation and thrombosis are of key interest
in cardiovascular research. Accordingly, recent evidence has highlighted that FXIIa
plays an important role in mediating pathological thrombosis, in addition to possessing
several previously unappreciated roles in regulating inflammation. Therefore, given
the importance of the thromboinflammatory process in contributing to atherogenesis
and AAA development, we investigated the efficacy of FXIIa inhibition as a potential
approach for prevention and treatment of atherosclerosis and AAA.
For AAA, there is increasing evidence that the thrombotic burden is associated with
the risk of aneurysm rupture. The highly proteolytic microenvironment of the thrombus
is proposed to decrease wall strength and thereby increase the risk of aneurysm rupture.[22]
[23]
[31] In our study, although similar numbers of animals developed aneurysms and IMTs,
the 3F7-treated cohort experienced less acute ruptures and developed smaller aneurysms
in comparison to the control mice. Upon histological examination of collagen deposition
within AAAs, an indicator of structural stability, we observed a thick, fibrous layer
of collagen deposited at the IMT–vessel interface in animals treated with 3F7. Recently,
a study by Moran et al[32] investigated the gene and protein profiles of both an ApoE−/− FXII−/− double-knockout and an ApoE−/− 3F7-treated mouse model. The authors reported decreases in ADAM-17, endothelial growth
factor receptor, and the matrix metalloproteinases MMP-2 and MMP-9 expression,[32] correlating to reduced local inflammation and remodeling, as well as to the preservation
of extracellular matrix integrity. As the luminal portion of the IMT is the most inflamed
area of an aneurysm, containing the highest proportion of proinflammatory infiltrating
cells,[33] the increased deposition of collagen at this interface is an exciting finding and
might contribute to the increased rate of survival associated with 3F7 administration.
3F7 significantly attenuated the development of atherosclerosis in ApoE−/− mice. Most strikingly, pharmacological inhibition of FXIIa prevented the development
of vulnerable atherosclerotic plaques in the TS model of plaque instability. Mice
treated with 3F7 exhibited significantly decreased necrotic core size and lipid deposition.
In accordance with our AAA data, we also observed marked increases in collagen deposition,
cap thickness, and the cap-to-core ratio. These are particularly important findings,
since it is well accepted that the extent of cross-linked collagen deposited within
the cap region is proportional to the stability of the plaque and increases in cap
thickness confer a more stable atherosclerotic phenotype.[34] Moreover, inhibition of FXIIa resulted in increases in intimal smooth-muscle cell
content and almost eliminated intraplaque hemorrhage, further highlighting the compelling
plaque-stabilizing effects of FXIIa inhibition. The remarkable magnitude of this plaque-stabilizing
effect achieved by 3F7 can also be seen in comparison to the effects seen with the
use of a myeloperoxidase inhibitor in the TS model, which despite reporting on only
small plaque-stabilizing effects attracted broad interest.[35]
Recent experimental data have highlighted the role of FXII/FXIIa in regulating inflammation,
including direct regulation of neutrophil function and wound healing[36] in addition to a central role in the pathogenesis of experimental autoimmune encephalomyelitis.[37] These findings have served to highlight that pharmacological inhibition of FXIIa
can achieve significant anti-inflammatory effects. In this regard, our findings that
3F7 administration significantly decreases VCAM-1 expression and CD68+ macrophage
infiltration point to a potential role of FXIIa inhibition as an anti-inflammatory
strategy for the prevention and potential treatment of atherosclerosis.
This concept is further supported by the fact that we observed downregulation of circulating
cytokines IL-1β, IL-12p40, and MCP-1. These cytokines and chemoattractants play important
roles in mediating monocyte/macrophage recruitment and differentiation into proinflammatory
M1 macrophages, and, specifically for IL-1β, foam cell apoptosis,[38] and likely help explain the reduction in macrophage infiltration and overall disease
attenuation. While the present study did not specifically examine the downstream signaling
effects of FXIIa, our data are consistent with a recent report demonstrating that
genetic deficiency of FXII afforded ApoE−/− mice protection from atherosclerosis.[6] Here, FXIIa was shown to stimulate the secretion of proinflammatory cytokines, including
IL-1β and IL-12, from bone marrow–derived macrophages and antigen-presenting cells,
which likely explains the beneficial anti-inflammatory effects of FXIIa inhibition.
It is important to note that another important function of FXIIa relates to its ability
to activate the kallikrein/kinin system (KKS) to yield the proinflammatory oligopeptide
BK. However, we found no detectable differences in plasma BK levels between 3F7 and
BM4-treated mice, suggesting that the proinflammatory effects of FXIIa in the context
of atherosclerosis are either largely localized to the site of the lesion and any
BK generated not detectable systemically, or independent of the KKS.
An outstanding issue pertaining to the role of FXII in mediating atherosclerosis relates
to what activates FXII within the confines of an atherosclerotic plaque. While this
was not a focus of our current study, it is likely that the activation of FXII in
atherosclerotic plaques is linked to the multitude of physiological FXII activators
that have been previously demonstrated to be abundant within atherosclerotic plaques.
Indeed, extracellular traps, misfolded protein aggregates, and activated platelets
have all been previously demonstrated to be present within plaques and are well-described
physiological activators of FXII.[39] Further emphasizing the central role of FXII in mediating atherothrombosis, deficiency
of FXII or FXIIa inhibition has been demonstrated to diminish thrombus formation on
atherosclerotic plaque material ex vivo.[40] Moreover, a recent study investigating pharmacological inhibition of FXI not only
implicated FXI/FXIa in the development of atherosclerosis and thereby indirectly supporting
the pathological role of FXIIa within the plaque confines,[41] but also provides further evidence that therapeutic targeting of coagulation factors
holds immense potential to impact cardiovascular clinical outcomes.
The TS mouse model representing unstable plaques as seen in patients is a unique preclinical
tool both to develop diagnostic approaches for the detection of unstable plaques and
for the development and testing of drugs for plaque-stabilizing effects.[42] For the latter, the promising effects of FXIIa inhibition hold great promise for
clinical translation, particularly as first clinical applications, utilizing the anti-inflammatory
and antithrombotic effects of FXIIa inhibition are currently trialed. However, our
data also indicate that FXIIa targeting can be used for diagnostic approaches. Anti-FXIIa
imaging was shown to be a potential means to identify unstable atherosclerotic plaques,
which is a long sought-after diagnostic approach with the strong translational perspective
of identifying patients at risk and ultimately preventing myocardial infarctions.
Together, our data demonstrate that inhibition of FXIIa impedes the development of
atherosclerosis and stabilizes vulnerable atherosclerotic plaques, in addition to
preventing AAA rupture in a process linked to its anti-inflammatory and stabilizing
effects. These data, coupled with the antithrombotic benefits of FXIIa inhibition
without interference with normal hemostasis, identify FXIIa inhibition as a potential
novel preventative and therapeutic strategy for unstable atherosclerosis and AAA.
However, our study does have some limitations. While we show for the first time that
the specific pharmacological targeting of FXIIa with 3F7 attenuated disease severity,
any contributions of zymogen FXII to the observed pathology cannot be ascertained
given the specificity of 3F7 for activated forms of FXIIa. As zymogen FXII is reported
to directly influence innate immune functions and exerts mitogenic activity in endothelial
and smooth muscle cells,[14] further studies are required to dissect the contributions of zymogen and activated
forms of FXII. In addition, we used only male mice for our investigations which, although
these diseases disproportionally affect males, may have led us to miss potential sex-specific
effects of FXIIa inhibition. Follow-up studies will need to include female mice to
exclude sex differences. Finally, our experimental models are designed to test for
prophylactic benefits of 3F7 in developing AAA and atherosclerosis. Further investigations
are required to establish the efficacy of 3F7 as a truly therapeutic approach. This
includes ascertaining the optimal dose and administration interval.
Conclusion
We demonstrate that 3F7 administration results in the stabilization of both AAAs and
vulnerable atherosclerotic plaques. 3F7 restricts the development of large aneurysms
and results in a lower incidence of acute aortic rupture, potentially a result of
the demonstrated increased collagen deposition between the IMT and the vessel lumen.
In unstable atherosclerosis, we observed less unstable plaque development with 3F7
administration, including increased collagen and smooth-muscle cell density. This
finding was accompanied by significant reductions in both systemic and local proinflammatory
markers. The ability of 3F7 to prevent pathological thrombosis and reduce inflammation
without impeding hemostasis is an additional unique and supportive feature of this
potential therapeutic approach. Our preclinical data indicate that FXIIa inhibition
has the potential to stabilize and prevent the rupture of AAAs and of vulnerable atherosclerotic
plaques. Clinical trials are warranted to demonstrate the translatability of our preclinical
data to clinically meaningful benefits in patients with AAA and atherosclerosis.
What is known about the topic?
-
Coronary atherosclerosis and abdominal aortic aneurysm and their resulting complications
of myocardial infarction and aortic rupture, respectively, are globally dominant causes
of mortality and morbidity.
-
Medical therapies that can prevent and stabilize vulnerable atherosclerotic plaques,
and rupture-prone AAAs are highly sought-after.
-
Activated coagulation factor XII (FXIIa) sits at the interface of both coagulation
and inflammation.
What does this paper add?
-
Inhibition of FXIIa via a monoclonal antibody reduces the size and increases the stability
of atherosclerotic plaques and abdominal aortic aneurysms.
-
FXII inhibition via 3F7 decreases markers of local and systemic inflammation in a
mouse model of vulnerable plaque.
-
3F7 mAb and its derivatives warrant further testing as potential drug candidates for
the prevention of myocardial infarction and the development and rupture of abdominal
aortic aneurysms.
