Abstract
Diabetes is a chronic metabolic disorder associated with the accelerated
development of macrovascular (atherosclerosis, coronary artery disease)
and microvascular complications (nephropathy, retinopathy and
neuropathy), which remain the principal cause of mortality and morbidity
in this population. Current understanding of cellular and molecular
pathways of diabetes-driven vascular complications as well as
therapeutic interventions have arisen from studying disease pathogenesis
in animal models. Diabetes-associated vascular complications are
multi-faceted, involving the interaction between various cellular and
molecular pathways. Thus, the choice of an appropriate animal model to
study vascular pathogenesis is important in our quest to identify
innovative and mechanism-based targeted therapies to reduce the burden
of diabetic complications. Herein, we provide up-to-date information on
available mouse models of both Type 1 and Type 2 diabetic vascular
complications as well as experimental analysis and research outputs.
Introduction
Diabetes Mellitus (DM), either Type1 (insulin-deficient) or Type2 (adult
onset, insulin resistance), is a chronic metabolic disorder that has
serious implications on cardiovascular health. Indeed, macro- and
microvascular complications associated with diabetes account for
approximately 65% of deaths in the diabetic population[1, 2].
Macrovascular complications associated with diabetes are mainly
represented by atherosclerosis and/or hypertension and their sequelae,
which include heart attacks and stroke, whilst microvascular
complications include chronic kidney disease, retinopathy and
neuropathy. The increased risk of cardiovascular complications in
diabetes is attributed to the elevated blood glucose levels and the
interplay between different cellular mechanisms in the disease setting
as a consequence of hyperglycemia[3]. These include, but are not
limited to, genetic, epigenetic, and cellular signalling pathways,
particularly the interplay between oxidative stress and inflammation,
which then result in imbalances in cellular metabolism contributing to
the development of cardiovascular complications.
Current standard of care for diabetic patients focusses on
glucose-lowering, lipid lowering and blood pressure control, however
these treatment options have yielded suboptimal outcomes on
cardiovascular end points, with the development of tolerance and
resistance to these therapies, particularly with respect to blood
pressure lowing medications limiting their long-term use[4, 5].
Newer, more innovative CV-specific drug treatments are urgently needed
to prevent diabetes-mediated vascular diseases. In this regard, animal
models have proven to be an invaluable tool in exploring and
characterising novel pathophysiological pathways, identifying
therapeutic targets and evaluating their in vivo potential.
However, it is critical to choose appropriate animal models that most
closely mimic the human pathophysiology of diabetes and its sequalae
such as the cardiovascular complications, to investigate potential
treatments and therapeutic strategies. To date, there is no perfect
animal model that encompasses all the pathophysiology and
characteristics of human disease but with advances in genetic
engineering and chemical manipulation, rodents have proven to be the
most reproducible, effective and preferred animal model for studies of
diabetic vascular diseases, particularly with respect to emulating
specific conditions that occur in humans. In this review, we provide a
comprehensive and up-to-date analysis on proven animal models of
diabetes-associated vascular diseases, with a focus on the analysis and
experimental outputs that can be achieved with these models.
Animal models of diabetes-associated macrovascular diseases
Vascular pathophysiology: key features that need to be
represented in animal models
Atherosclerosis is the principal cause of death and disability in the
diabetic population, with the incidence of an atherosclerotic event
occurring approximately 14.6 years earlier in this patient cohort. The
pathophysiological events that lead to disease progression in the
presence of diabetes have been extensively reviewed[1, 6, 7],
however, key factors that are important to consider in choosing a
relevant animal model of diabetes–associated atherosclerosis will be
discussed. A main clinical feature that presents in uncontrolled or
poorly controlled Type1 and Type2 diabetic patients is hyperglycemia,
the key driver of vascular injury. Elevated blood glucose, primarily via
advanced glycation end products (AGEs) and their interaction with
receptor for advanced glycated end products (RAGE) drives
pro-inflammatory/pro-oxidant pathways to elicit molecular, cellular and
vascular injury[8, 9]. A further key feature of diabetes is
dyslipidemia manifested through elevated triglycerides, decreased high
density lipoproteins (HDL) and changes in low density lipoprotein (LDL)
structure to a more atherogenic profile[10], which results from the
ineffective clearance of post-prandial lipoprotein rather than increased
circulating levels of fasting lipoproteins[11]. In addition, the
heightened state of oxidative stress and inflammation perturbs cellular
metabolism particularly within endothelial cells which leads to
endothelial dysfunction. Together with a build-up of oxidised LDL
particles, endothelial dysfunction promotes increased adhesion and
infiltration of leukocytes into the vessel wall and the formation of
foam cells, thereby leading to the acceleration of atherosclerotic
processes[1].
Diabetes-associated atherosclerosis is most commonly studied in mice
despite the fact that humans and mice differ in certain characteristics
that affect the development of disease progression. Firstly, mice are
generally protected from atherogenesis due to differences in their
lipoprotein profile, with mice exhibiting elevated HDL levels due to the
absence of cholesteryl ester transfer protein (CETP)[12, 13].
Therefore, atherosclerotic plaque generation requires genetic
manipulation such as the deletion of the apolipoprotein E (ApoE) gene or
the LDL receptor (LDLR) gene together with dietary interventions.
Additionally, humans and mice have differences in cardiovascular anatomy
and haemodynamic physiological forces that influence the sites of
atherosclerosis[14]. Lesions predominantly occur in the coronary
arteries, carotid and peripheral vessels in humans whilst in mice,
lesions are commonly studied in the aortic sinus and the aortic
arch[14]. A summary of the models of diabetes-associated
atherosclerosis is shown in Figure 1.
Rodent models of diabetes-associated atherosclerosis2.2.1. Models of Type1 diabetes-associated atherosclerosis
To replicate human disease, most mouse models of Type1 diabetes are
insulin deficient, either by chemical induction or genetic manipulation.
The most extensively studied Type1 model of diabetes-associated
atherosclerosis is the streptozotocin (STZ) induction model in the ApoE
knockout (-/-) mouse, initially characterised by Park
et al[15]. A major advantage of the STZ-induced
ApoE-/- mouse model is the vast amount of published
literature available utilising this model allowing researchers to
compare and contrast data, cellular mechanisms and therapeutic
interventions. STZ, a pancreatic β-islet cell toxin, enters the cell via
Glut-2 transporters to mediate its cytotoxic effect, thereby limiting
insulin production, leading to hyperglycaemia[16]. Generally, STZ is
administered as five consecutive low doses (55mg/kg/day)[17-19] but
certain groups have demonstrated more consistent elevations in blood
glucose by using two consecutive high doses (100mg/kg/day)[20-24].
This becomes particularly important for longer term studies where blood
glucose levels may decline due to recovery of β-islet cells. Despite the
benefits of a robust and consistent increase in hyperglycemia of around
25-30mmol/L, there are limitations that may preclude the use of STZ
under certain circumstances. For example, STZ at high doses has been
shown to affect the function of other cells expressing the Glut-2
transporter, such as hepatocytes and epithelial cells of the renal
tubules, which may be exposed to the toxic action of STZ leading to
unwanted side effects in the liver and kidneys[25]. In addition, a
further limitation of using STZ as the chemical inducer of diabetes is
that females, particularly mice, have been shown to be resistant to STZ,
often requiring higher doses of STZ to obtain equivalent levels of
hyperglycemia compared to male counterparts[26]. Thus, female mice
are commonly omitting in pre-clinical research for diabetic
complications when STZ is the preferred method of Type1 diabetes
induction. STZ also exhibits broad spectrum antibacterial properties
which may alter the gut microbiota[27].
Despite the noted limitations, the STZ-induced diabetic
ApoE-/- mouse model is the preferred model for
studying atherosclerosis, since these mice exhibit hyperglycemia as well
as dyslipidemia, as indicated by the elevated blood glucose levels,
total cholesterol, LDL and triglyceride levels. Plaque formation is
increased 3-6 fold in this model over a 10-20 week period, with early
discrete lesions observed within the aortic sinus and the aortic arch;
however, over a longer course of diabetes, lesions additionally develop
over the thoracic and abdominal aortic regions. This model has been
associated with endothelial dysfunction, increased inflammation and
oxidative stress as well as macrophage foam cell formation, all of which
are hallmarks of atherosclerotic pathogenesis. More importantly, the
STZ-induced ApoE-/- mouse model has been particularly
useful in investigating potential therapies, such as but not limited to
soluble RAGE (sRAGE), peroxisome proliferator-activator gamma agonists,
anti-oxidants (ebselen, dimethyl furate), SGLT2 inhibitors and ApoA1
mimetics, in their ability to attenuate diabetes-associated
atherosclerosis, independent of blood glucose lowering effects [21,
22, 28-32]. As the ApoE-/- mouse model is well
established and easy to breed, cross-breeding ApoE-/-mice with mice harbouring genetically altered proteins such as
transcription factors, receptors and/or enzymes enables the elucidation
of their role in diabetic atherosclerotic mechanisms. On the contrary,
STZ-induced diabetic LDLR-/- mice do not exhibit
extensive atherosclerotic lesions despite having hyperglycemia and
elevated LDL/vLDL levels and often require additional diet
supplementation[33] making them a more ideal model of Type2
diabetes.
A less well-known model that is used in Type1 diabetic atherosclerotic
research, particularly if the side-effects of STZ are of concern (as
detailed above), is the spontaneous genetic Akita mouse, cross bred with
either the ApoE-/- or LDLR-/- mouse.
The Akita mouse, first selected in Japan, carries an autosomal dominant
missense mutation in the insulin2 gene, resulting in impaired proinsulin
processing and aggregation of misfolded insulin protein, rendering it
insulin deficient. Akita mice develop typical Type1 diabetes
characteristics, including hyperglycemia, polyuria and polydipsia, as
well as impaired vascular tone as early as 3-4 weeks of age, making it
an ideal model to study the effects of diabetes on endothelial
dysfunction and macrovascular complications[34]. When cross-bred to
the ApoE-/- or LDLR-/- mouse, this
model exhibits dyslipidemia, mainly manisfested as increases in
triglycleride and non-HDL cholesterol levels, and an approximately 2 to
3-fold increase in atherosclerotic lesions[35-37]. This is
accompanied by increased infiltration of immune cells into the plaque,
in particular macrophages and T-cells, as well as augmentation of
vascular inflammation as demonstrated by increased levels of TNF-α,
IL-1β and MCP-1, all of which are key mediators involved in plaque
progression[36, 37]. Interestingly, the differences in plaque size
are not as pronounced in female Akita/LDLR-/- mice as
they are in male mice, suggesting gender differences could play a part
in diabetic atherosclerosis in this model.
The models described thus far are promising to investigate the
mechanisms of endothelial dysfunction, vascular inflammation and plaque
formation in a diabetic setting, however, it is difficult to discern
whether these arise as a direct effect of the hyperglycemia or as a
consequence of other confounding atherogenic factors such as the
dyslipidemia under these conditions. To circumvent this problem,
Vikramadithyan et al have developed a mouse model to study the effects
of hyperglycemia in accelerating atherogenesis by manipulating Aldose
reductase, an enzyme involved in catalysing the reduction of glucose to
sorbitol, in combination with STZ[38]. Aldose reductase has been
shown to accelerate diabetic vascular complications through the
increased production of reactive oxygen species (ROS). Indeed,
overexpression of human aldose reductase in the diabetic
LDLR-/- mouse resulted in a 2-fold increase in lesion
size as compared to diabetic LDLR-/- mice alone,
driven by defects in glucose-driven ROS signalling[38]. Another
model developed to closely mimic the autoimmune destruction of
pancreatic β cells as observed in human Type1 diabetes is the induction
of lymphocytic choriomeningitis virus (LCMV) infection [39, 40]. The
LCMV glycoprotein (GP) transgene is controlled by the insulin promoter
in β cells, which is rapidly destroyed upon infection. These
LDLR-/- mouse injected with the LCMV GP developed
severe hyperglycemia that was associated with accelerated lesion
formation in the absence of hypertriglyceridemia[39, 40]. Moreover,
atherosclerotic lesions were morphologically similar to human lesions
particularly with increased arterial macrophage accumulation and
intralesional haemorrhage[39]. Nonetheless, further investigation is
required to delineate the exact role of altered lipoprotein profiles and
hyperglycemia in preclinical animal models of Type1 diabetes-induced
atherosclerosis.