RNAi therapy to the wall of arteries and veins: anatomical, physiologic, and pharmacological considerations

Anatomical and physiological differences of vascular conduits and disease

The anatomy of arteries and veins reveals similarities yet also significant histological differences. The inner lining of arteries and veins (tunica intima) consists of a single layer of EC seated on a basement membrane (BM). The layers of the BM are composed of an intricate network consisting of various collagens types, glycoproteins and proteoglycans, and cell–cell, and cell–matrix interaction regulators such as integrins. Different from sinuses in the liver and spleen, which have a discontinuous BM and therefore promote extravasation of RNAi, the BM of large arteries and veins is continuous and provides an anatomical barrier. The BM is connected to a thin layer of subendothelial connective tissue. Different from veins, arteries and arterioles also have as the outer margin of the tunica intima a prominent internal elastic membrane (lamina) with elastin containing fibers. This layer presents another physical barrier for RNAi for transmural delivery.

The next layer, tunica media is mostly comprised of circularly arranged smooth muscle cells with interspersed reticular and elastic fibers. This layer’s thickness varies significantly between veins and arteries. Naturally a thicker layer of SMC in larger arteries provides a greater challenge for a homogenous, transmural transfection when compared to a thin-walled vein graft. As mentioned before, VSMC appear less susceptible to RNAi compared to EC [9–12]. If VSMC are the designated primary target cells and in the absence of a technology to achieve VSMC-specific delivery, it is possible that a disproportionate amount of RNAi oligomers will transfect nearby EC. This needs to be factored into the silencing strategy as it could cause deleterious effects. This is especially the case if regulators of apoptosis, cell cycle, or migration are targeted [13–15].

The outer most layer, tunica adventitia is made up of connective tissue. In large arteries an external elastic membrane may separate the tunica adventitia from the tunica media. Large arteries and veins receive additional blood supply via vasa vasorum, which enter from the adventitial site. Given the low oxygen content of venous blood, it is conceivable that large veins are more dependent on vasa vasorum for their nutrient/oxygen supply than arteries. In the setting of vein graft implantation, that supply route may be disrupted. This may contribute to the formation of IH. It is also believed that vasa vasorum play a significant role in atherosclerosis progression [16].

Interestingly, vasa vasorum appear to cease to supply oxygenated blood to the arterial wall at the level of the renal arteries, which may help explain an increased incidence of aortic aneurysms in this region [17]. The expression of endothelial alpha(v)beta3 integrin was used to pharmacologically target vasa vasorum and thus modulate atherosclerosis [16].

Atherosclerosis

Atherosclerosis (ATH) by some is considered a chronic inflammatory condition that involves a complex interplay of immune and stromal cells, cytokines and enzymes. It is characterized by a disturbed loco-regional lipid metabolism, extracellular matrix organization, and cellular homeostasis leading to lipid plaque build-up and ultimately risk for plaque rupture and vascular narrowing or occlusion [8, 18]. Hyperglycemia is further believed to induce VSMC resistance to apoptosis and thus contributing to diabetic vasculopathy [19]. Multiple potential targets for a siRNA therapy of ATH, AA, and IH have been identified, yet clinically convincing evidence of an efficacious therapy is lacking [7, 8].

A recent review from Welten et al. dissects the contribution of various miRNA clusters to the maladaptive processes of atherosclerosis and restenosis [20]. Prominent miRNA amongst those are miR-126, miR-155, the miRNA gene clusters 17-92, and miRNA 23/24/27, 143/145 [20]. Additionally, transcoronary gradients of anti-atherosclerotic miR-126-3p and miR-145-5p were found to correlate with the extent of thin cap fibroatheromas [21]. miRNA-210 is believed to enhance fibrous cap stability in atherosclerotic lesions [22]. miRNA targets for atherosclerosis were also recently reviewed elsewhere [23].

Intimal hyperplasia (IH)

In the case of IH in the setting of vein graft surgery and vascular implantation injury, synthetic VSMC migrate towards the lumen of the vessel. There, VSMC proliferate and secrete pro-inflammatory cytokines and extracellular matrix proteins forming the bulk of the eventual IH lesion [7].

miRNA have also shown to be involved in IH formation [24, 25]. One of the genes that has been shown to regulate the expression of multiple miRNA in VSMC is Thrombospondin 1 (TSP-1), a glycoprotein involved in cell–cell and cell–matrix contacts [25]. The most upregulated miRNA in response to TSP-1 was miR-512-3p, while the most downregulated miRNA was miR-25-5p. Interestingly, five members of the mir-17-92 cluster were downregulated [25].

Some strategies to attenuate IH aimed at attenuating VSMC proliferation after vascular injury [13, 26, 27]. While this seems to make physiologic sense, clinically the initial results have been disappointing. The largest, prospective, randomized controlled trial in humans to date (PREVENT III) designed to attenuate vein graft failure by use of oligonucleotide anti-sense therapy targeting E2F, a transcription factor promoting G1/M transition, failed to show protection from IH [26, 27]. There has been a lot of debate as to why to the PREVENT III trial failed. Be it the degree or duration of gene silencing, or the target E2F itself. Another consideration is that EC are more susceptible to RNAi than VSMC under identical conditions [9–12]. Hence, it is conceivable, that bystander EC were transfected and their altered ability to proliferate in the setting of vascular injury may have contributed to the trial’s failure.

Distinct gene expression and stress response patterns of VSMC and EC from different vascular beds

Not surprisingly, relevant differences exist amongst the same cell type (EC or VSMC) within the separate vascular beds. A study comparing canine VSMCs within the aorta (Ao), branch pulmonary artery (bPA), main pulmonary artery (mPA) and inferior vena cava (IVC) revealed not only obvious histologic differences in layer thickness and cellular organization, but also significant protein expression differences with regards to smooth muscle actin (SMA), myosin heavy chain (MHC) and smooth muscle myosin heavy chain isoform 2 (SM2) [30].

Separately, differential expression patterns were found with regards to Hox4. Products of the hox gene family regulate the cranio-caudad organization of the body during embryogenesis. Differential Hox4 expression has been found along the aorta in EC and VSMC of baboons. Thoracic EC and VSMC revealed higher Hox4 levels compared to corresponding abdominal aortic cells [31]. Further, human AAA specimens were found to have a significantly lower Hox4 expression when compared to healthy controls [31].

Another example is the protein testin, which was found to be differentially expressed in coronary and internal mammary arteries. Testin is a cytoskeleton-associated protein that localizes along actin stress fibers, at cell–cell-contact areas, and at focal adhesion plaques. Testin was also found to have tumor suppressor gene function and may be relevant for EC function [32, 33]. Testin silencing in EC promoted oxidized-LDL-mediated monocyte adhesion to ECs, EC migration and the transendothelial migration of monocytes, while overexpression of testin mitigated these effects [33].

Aside from varying intrinsic protein expression profiles it could be shown that VSMC from different segments of the circulation respond distinctly to noxious stimuli. Oxidized low-density lipoprotein (OxLDL) treatment in saphenous vein graft VSMC (SV-VSMC) led to an increase in proliferation and migration as well as NF-kappa B activation. In contrast, OxLDL inhibited proliferation and migration in coronary VSMC. In addition, significant differences in cytokine, chemokine extracellular matrix protein expression were noted between these cell groups [34].

In line with this are observations derived from in vitro experiments, which revealed that SV-VSMC were found to be significantly more proliferative and displayed a more migratory and invasive behavior than internal mammary artery VSMC [35].

Yet in contrast, human VSMC derived from atherosclerotic lesions of infragenicular arteries displayed significantly increased rates of proliferation, adhesion, and migration as compared to human saphenous vein VSMC [36].

Also, the response to hypoxia appears to be different in arterial and venous SMC. While both cell fractions showed a decreased proliferative response to hypoxia, arterial SMC showed a significant upregulation of vascular endothelial growth factor (VEGF-A), while venous SMC did not. VEGF-R 2 expression was found to be upregulated in hypoxic venous SMC, but not in arterial SMC [37]. The same investigators hypothesized that VEGF-A may be a target gene of mir-125b, 29a, and 29b. Interestingly, hypoxic conditions induced a 15-fold increase of mir-125b in VSMC compared with ASMC, which did not show a significant change. In the author’s opinion this could explain the lack of VEGF-A upregulation in hypoxic venous SMC [37]. Other relevant miRNA include miR-143 and 145, which appear to have significant roles in SMC fate and plasticity [38, 39]. Adding complexity to the VSMC-EC interaction is the existence of intercellular nanotubes that shuttle VSMC derived miR-143 and 145 to EC and thereby influence EC biology [40]. This mechanism, at least in part, was triggered by TGFβ and vessel stress [40].

Further complicating is the finding that significant differences between EC from various vascular beds exist [41]. There appear to be even differences in the phenotypes of aortic valve EC on the ventricular site when compared to the aortic site. In vitro shear-stress experiments revealed that waveforms simulating the ventricular flow pattern selectively upregulated the atheroprotective transcription factor Kruppel-like factor 2 (KLF2), while suppressing the pro-inflammatory chemokine monocyte-chemotactic protein-1 (MCP-1) in EC [42].

Separately, bone morphogenetic protein (BMP) signaling has been found relevant for a variety of pathways involved in EC biology. Heterogeneous transcriptional activity seemed to correlate with vasculature undergoing hemodynamic alterations [43]. BMP signaling is connected to other major signaling pathways including Notch, WNT, and is altered in response to hypoxia [44].

Locoregional differences in miRNA expression patterns as a function of flow disturbances have been investigated, coining the term “flow-sensitive miRNA” or “mechano-miRNA” [45]. miRNAs such as, miR-10a, miR-19a, miR-23b, miR-17-92, miR-21, miR-663, miR-92a, miR-143/145, miR-101, miR-126, miR-712, miR-205, and miR-155 are counted amongst those mechano-miRs [45].

In addition, there appear to be differences in siRNA susceptibility between EC and VSMC and possibly even between the various VSMC fractions under identical transfection conditions [9, 10].

All these biological differences help explain the varying loco-regional responses to vascular injury including atherosclerosis, postangioplasty restenosis and vein graft disease [34]. These data underscore the importance of a comprehensive characterization of the specific target cell subtype and the spatial differences with the cell subtype. Further, it is important to understand the pathophysiologic consequences that a specific intervention inflicts on these cells. This includes the effects of RNAi therapy itself including the effects of the transfection reagents used.

RNAi delivery methods

Systemic, naked siRNA administration without technology to target specific cells types leads to renal siRNA secretion within 20 min as well as rapid plasma nuclease degradation. In addition, significant degradation and phagocytosis via the reticuloendothelial system occurs which also shortens the siRNA’s systemic half-life [46].

In parallel, several chemical modifications to the RNA have been introduced including locked nucleic acid technology (LNA), in which an additional covalent bond within the RNA backbone is formed. This has proven to improve stability to the RNA molecule and protection from phagocytosis [47, 48].

Liposomes are spheres consisting of customizable phospholipid layers that can be used for drug delivery. Liposomes widely used to facilitate diagnostic and experimental siRNA silencing vitro and are one of the most commonly used transfection modality [9, 49, 50]. However, liposomal siRNA formulations also undergo significant uptake by macrophages, liver and spleen (RES) limiting the ability to successfully reach the target genes in vivo. In addition, problems with toxicity, immune response, non-specific uptake, and the risk of off-side effects have been raised [9, 49, 51, 52].

The introduction of poly(ethylene glycol) (PEGylation) into liposomal formulations is another commonly performed process aimed at improving drug delivery efficacy. PEGylation is thought to confer some protection from mononuclear phagocytosis through the creation of a protective hydrophilic film on the surface of the liposome and decrease renal clearance due to a size increase of the liposomal complex [53–57].

Cationic polymers such as polyethyleneimine (PEI) can also be used to complex RNA complexes and improve transfection efficiency into vascular cells in vitro and in vivo [28, 58–60]. The positive surface charge of PEI-siRNA complexes may be utilized for adsorption onto vascular materials [58]. Incorporation of albumin into PEI-siRNA complexes provided protection from extracellular endonucleases and improved internalization and silencing efficiency in vitro [61]. However, PEI has been shown to have significant impact on global gene expression, leading to changes in 213 genes in human aortic SMC. These genes were mostly related to inflammation and immune response. These findings illustrate how PEI alone could affect the results of the RNAi therapy [29].

Another promising modality is RNAi containing nanoparticles. Some of these nanoparticles have self-assembling properties. The individual composition of these nanoparticles is highly customizable and can be adjusted to the specific needs of the therapy approach [62–66]. These nanoparticles can consist of degradable, biologically compatible materials such as poly(lactide-c-glycolide) (PLGA) [67]. The polymer’s degradation profile might allow for sustained RNAi release for vascular delivery [68, 69]. Polymer blends and co-polymers may also facilitate sequential RNAi release.

RNAi encoded in lentiviral and adenoviral vectors was successfully delivered to cardiovascular cells and other tissues [13, 65, 70–74]. Due to concerns of immunogenicity associated with older adenoviral constructs and possible insertional mutagenesis by lentiviruses, novel adenovirus associated viruses (AAV) have been developed. AAV have shown some promise for anti-angiogenic therapies for human retinal diseases [75–77]. While AAV is a powerful tool, there is ongoing research to develop modified AAV surface capsids for cell-specific RNAi delivery [78].

Many reviews exist that provide comprehensive overviews of RNAi vectors, chemical modifications and transfection agents including detailed capabilities and current limitations of either technology [50, 79, 80]. For the purpose of this review we will focus on approaches pertaining to vascular RNAi delivery.

Results of vascular RNAi therapy for vascular injury remodeling

Target choice

Vascular injury is characterized by a dramatic upregulation of thousands of genes within a matter of hours as demonstrated in a study of vein graft implantation in canine [106]. Over the following weeks these gene expression changes largely approach baseline [106]. Despite this apparent gene expression “near-normalization” after 4 weeks, the process of vascular remodeling and thus the growth of vascular lesions continues past that point. It is therefore of interest to have the option of a sustained release of RNAi to the vascular wall to change gene expression past the immediate period of graft implantation or endovascular intervention. Analysis of gene networks revealed significant signaling redundancy, which suggests it might be prudent to silence multiple genes simultaneously and/or sequentially in order to optimize the clinical effect of siRNA therapy. This became evident in a recent rat carotid artery angioplasty model, in which the extracellular matrix protein thrombospondin-2 was silenced through single intravascular siRNA administration. Anti-TSP-2 siRNA complexed with PEI resulted in TSP-2 protein level suppression for at least 21 days. This resulted in changes in downstream transforming growth factor–beta (TGF-β) and matrix metalloproteinase-9 (MMP-9) signaling and increased M2 macrophage polarization. However, no significant changes in the intima/media ratios were noted [59]. This data underscores the importance of target gene selection. While in this case, TSP-2 silencing yielded the anticipated downstream signaling effect and even a desirable macrophage phenotype switch, it did not result in a histologically relevant effect.

Given the discussed issues of cell specific RNAi delivery, target gene choice becomes a conundrum. Silencing of the target gene should result in a robust alteration in the biology of the targeted cell. In contrast, inadvertent target gene silencing in bystander cells should not impede those cells’ inherent biological activities. For example, the cell cycle and apoptosis regulator survivin (SVV) is highly upregulated in SV-VSMC the setting of IH [13, 107]. This appears to promote the proliferative, apoptosis resistant and migratory VSMC phenotype responsible for IH. In vitro silencing of SVV in SV-VSMC resulted in a cell cycle block with a subsequent decrease in proliferation. While it did not increase the rate of apoptosis in SV-VSMC to noxious stimuli, it did impair SV-VSMC migration.

In contrast, it has been shown, that overexpression of SVV in EC resulted in increased viability and migratory capability, but reduced apoptosis. SVV overexpressing EC were also associated with higher levels of angiogenesis in vivo [13, 107–110].

One of those candidate genes that differentially affects biology of EC and VSMC may be MARCKS (myristoylated alanine rich protein kinase C substrate) [111–114]. MARCKS silencing in VSMC attenuated proliferation and migration, while in EC these two cell functions appeared unaltered [114]. This of course does not preclude, that other EC functions may be affected by MARCKS silencing that could be relevant for vascular injury remodeling.

Conceptually an intriguing group of target genes are inflammatory cytokines and extracellular matrix proteins that are secreted by both, EC and VSMC. Arguably, silencing these targets in both cell fractions may prove beneficial.