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Neuroprotection in early stages of Alzheimer’s disease is promoted by transthyretin angiogenic properties
Alzheimer's Research & Therapy volume 13, Article number: 143 (2021)
Abstract
Background
While still controversial, it has been demonstrated that vascular defects can precede the onset of other AD hallmarks features, making it an important therapeutic target. Given that the protein transthyretin (TTR) has been established as neuroprotective in AD, here we investigated the influence of TTR in the vasculature.
Methods
We evaluated the thickness of the basement membrane and the length of brain microvessels, by immunohistochemistry, in AβPPswe/PS1A246E (AD) transgenic mice and non-transgenic mice (NT) bearing one (TTR+/−) or two (TTR+/+) copies of the TTR gene. The angiogenic potential of TTR was evaluated in vitro using the tube formation assay, and in vivo using the chick chorioallantoic membrane (CAM) assay.
Results
AD transgenic mice with TTR genetic reduction, AD/TTR+/−, exhibited a thicker BM in brain microvessels and decreased vessel length than animals with normal TTR levels, AD/TTR+/+. Further in vivo investigation, using the CAM assay, revealed that TTR is a pro-angiogenic molecule, and the neovessels formed are functional. Also, TTR increased the expression of key angiogenic molecules such as proteins interleukins 6 and 8, angiopoietin 2, and vascular endothelial growth factor, by endothelial cells, in vitro, under tube formation conditions. We showed that while TTR reduction also leads to a thicker BM in NT mice, this effect is more pronounced in AD mice than in NT animals, strengthening the idea that TTR is a neuroprotective protein. We also studied the effect of TTR tetrameric stabilization on BM thickness, showing that AD mice treated with the TTR tetrameric stabilizer iododiflunisal (IDIF) displayed a significant reduction of BM thickness and increased vessel length, when compared to non-treated littermates.
Conclusion
Our in vivo results demonstrate the involvement of TTR in angiogenesis, particularly as a modulator of vascular alterations occurring in AD. Since TTR is decreased early in AD, its tetrameric stabilization can represent a therapeutic avenue for the early treatment of AD through the maintenance of the vascular structure.
Introduction
Alzheimer’s disease (AD) patients undergo several neurovascular changes at different levels. Brain vascular dysregulation is the earliest and strongest factor during disease progression and is followed by amyloid-β (Aβ) peptide deposition, glucose metabolism dysregulation, functional impairment, and gray matter atrophy, in this order [1]. Decreased expression of the low-density lipoprotein receptor-related protein 1 (LRP-1) and P-glycoprotein (P-gp), as well as upregulation of the receptor for advanced glycation end products (RAGE), are mechanisms reported to be changed in AD patients, leading to Aβ accumulation in the brain [2, 3]. In addition to defective clearance mechanisms, increased endothelial pinocytosis, decreased number of mitochondria, decreased glucose transporter (GLUT)-1, and loss of tight/adherents junctions are features detected in AD [4]. The reduction of the capillary density is also characteristic of the AD brains [5]. This is due to an aberrant angiogenesis with premature pruning of capillary networks. This defective angiogenesis may be caused by a lack of angiogenic stimuli and unresponsive endothelium [6]. Although other authors describe increased vascular density in AD [7], the underlying angiogenic process has pathological characteristics. Some studies suggest that the promotion of angiogenesis results in concomitant blood-brain barrier (BBB) disruption and vessel leakiness [7]. Other studies defend that the vascular damage is a consequence of poor blood perfusion of the brain, leading to hypoperfusion/hypoxia causing the BBB dysfunction [8]. Other authors argue that the accumulation of Aβ in the walls of the capillaries can contribute to the reduced brain capillary density in AD via anti-angiogenic activity [9, 10]. Another observed alteration in AD is the increased thickness of the vascular BM in AD [11]. Since the increase in BM thickness occurs before Aβ deposition, it is speculated that it functions as a physical barrier to the Aβ clearance across the BBB [12]. Some studies have related this BM thickening with increased collagen IV content, in AD and aging [13, 14].
Transthyretin (TTR), a 55-kDa homotetrameric plasma and cerebrospinal fluid (CSF) protein, transports retinol through binding to the retinol-binding protein (RBP), which binds at the surface of TTR, and thyroxine (T4), which binds at a central hydrophobic channel formed at the dimer-dimer interface [15]. In the CSF, TTR is the main Aβ binding protein [16], providing neuroprotection by avoiding Aβ aggregation [16,17,18,19,20,21,22,23] and toxicity [17, 24], and by participating in Aβ brain efflux at the BBB [25]. TTR is early decreased in AD, both in plasma [26,27,28] and in the CSF [29], probably due to its tetrameric instability [26, 30], hypothesized to result in accelerated clearance and lower levels. TTR instability is also a key feature in familial amyloid polyneuropathy (FAP), a systemic amyloidosis that is usually caused by mutations in TTR. The amyloidogenic potential of the TTR variants is inversely correlated with its tetrameric stability [31], and the dissociation of the tetramer into monomers is at the basis of the events that culminate with TTR amyloid formation [32, 33]. TTR stabilization, used as a therapy in FAP [34, 35], can be achieved through the use of small-molecule compounds sharing molecular structural similarities with T4 and binding in the T4 central binding channel [36,37,38]. Although no TTR mutations have been found in AD patients [22], TTR stabilization has also been proposed as a therapeutic strategy to recover its ability to protect in AD [19, 39], and shown beneficial in a mouse model of AD [39, 40]. Iododiflunisal (IDIF), a potent TTR stabilizer, was administered to AD mice and bound plasma TTR displacing T4, resulting in decreased Aβ amyloid burden and total Aβ brain levels, and improved cognition [40]. Interestingly, TTR stabilization by IDIF improves TTR-assisted Aβ brain efflux in vitro and enhanced the expression of LRP-1 in vivo [30]. The formation of TTR-IDIF complexes enhances BBB permeability of both IDIF and TTR, in vivo [41].
TTR has also been implicated in angiogenesis and the first reports of its involvement have been described in diseases such as FAP [42]; in diabetic retinopathy (DR) [43, 44], and lately, in cancer [45]. As reported, a study investigated the effect of TTR in angiogenesis by treating human umbilical vein endothelial cells (HUVECs) with wild-type (WT) TTR or a common FAP TTR mutant, V30M. The authors concluded that the TTR mutant inhibited cell migration and decreased survival relative to the WT TTR, by down-regulating several pro-angiogenic genes for angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF) receptors 1 and 2, basic fibroblast growth factor (bFGF), and transforming growth factor-beta 2 (TGF-β2) [42]. In another study, to investigate how TTR affects the development of new vessels in DR, human retinal microvascular endothelial cells (hRECs) were cultured with TTR in natural and simulated DR environments (hyperglycemia and hypoxia). In the DR environment, TTR inhibited cell proliferation, migration, and tube formation, by repressing the expression of the pro-angiogenic genes Ang-2 and VEGF receptors 1 and 2 [43]. Conversely, in a low glucose environment, these angiogenesis-related features were improved by TTR. Recently, it was reported that TTR levels were increased in human serum of lung cancer patients. Additionally, TTR was shown able to promote tumor growth by enhancing several lung ECs functions as permeability, migration, and tube formation [45]. However, TTR potential in angiogenesis has never been addressed in vivo and the possible participation of TTR in brain angiogenesis and vascular alterations has never been elucidated.
Taking these evidences into account, this work aimed at investigating the angiogenic potential of TTR and at assessing its involvement in the vascular impairment that occurs in AD.
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