The anatomical sources of neointimal cells in the arteriovenous fistula

Abstract

Neointimal cells are an elusive population with ambiguous origins, functions, and states of differentiation. Expansion of the venous intima in arteriovenous fistula (AVF) is one of the most prominent remodeling processes in the wall after access creation. However, most of the current knowledge about neointimal cells in AVFs comes from extrapolations from the arterial neointima in non-AVF systems. Understanding the origin of neointimal cells in fistulas may have important implications for the design and effective delivery of therapies aimed to decrease intimal hyperplasia (IH). In addition, a broader knowledge of cellular dynamics during postoperative remodeling of the AVF may help clarify other transformation processes in the wall that combined with IH determine the successful remodeling or failure of the access. In this review, we discuss the possible anatomical sources of neointimal cells in AVFs and their relative contribution to intimal expansion.

Introduction

The mature arteriovenous fistula (AVF) is the preferred vascular access for hemodialysis given its improved performance and lower complications rates compared to prosthetic grafts or central venous catheters.^1,2 While complications are less frequent in AVFs than in other vascular accesses, early and late AVF failure still remain major morbidities for dialysis patients, and important contributors to hemodialysis cost.^3,4 Intimal hyperplasia (IH) is the landmark histological feature in both the pre-access vein and the AVF.^5,6 The rapid expansion of the intima following AVF creation may contribute to the process of non-maturation⁷ while also play important roles in hemostasis. In fact, IH is present in both stenotic and non-stenotic areas of the AVF indicating a role beyond failure.⁸ Postoperative IH is a build-up of mostly myofibroblast-like cells (neointimal cells) and extracellular matrix (ECM) within the innermost layer of the fistula wall.^9,10 Occasionally, very few inflammatory cells and contractile smooth muscle cells (SMCs) are also found infiltrated in the intima.^6,11 The development of IH involves diverse and complex signaling cascades that ultimately result in intimal accumulation of myofibroblastic cells.^12,13 The origin of such cells has long been an issue of speculation and investigation. Identifying their sources is crucial to understand postoperative AVF remodeling and design better strategies for the prevention or treatment of AVF failure.

This review gives an overview of novel developments in the origin of cells populating the AVF intima (Figure 1). We assimilate, critique, and reconcile experimental and clinical evidence suggesting that neointimal cells within the fistula wall may derive from a variety of sources that include not only adventitial fibroblasts and SMCs, but also circulating and vascular wall “progenitor” cells. Of note, we limit our discussion to the evidence presented by human and experimental AVFs and avoid inferring conclusions from “apparently” related experimental models of arterial restenosis, atherosclerosis, and transplant arteriosclerosis.

Figure 1.

Potential anatomical sources of neointimal cells in the AVF.

The vein wall

Arteriovenous fistulas are created by end-to-side anastomosis of a selected vein with the adjacent artery, preferably using the cephalic or basilic veins in one of the upper extremities.¹⁴ These veins are formed by three anatomical layers: (1) the tunica intima, (2) the tunica media, and (3) the tunica adventitia (external).¹⁵ The intima is the innermost layer of the vein and is covered on the luminal side by a thin endothelial line that lies on a fenestrated basement membrane. The thickness and composition of the venous intima varies from thin and almost acellular to thick and rich in myofibroblasts and scattered SMCs.^16-18 The thickness of the intima in the pre-access vein neither predicts AVF stenosis nor failure.^19-21 The intima is surrounded by the media, which is mainly composed of circumferentially arranged and contractile SMCs interlaced with collagen and elastic fibrils. These contractile SMCs are recognized by their expression of smoothelin, smooth muscle myosin heavy chain (SM-MHC), and desmin.^17,22 The outer layer of the vein is the adventitia formed by a loose collagenous network interspersed with vasa vasorum. Abundant fibroblasts, immune cells, and longitudinal or spirally arranged contractile SMCs are also found in the adventitial zone adjacent to the media.¹⁸ Collagen fibers blend with the surrounding adipose tissue in the case of superficial, subcutaneous veins.

The AVF wall

The architecture of the pre-fistula vein dramatically changes after anastomosis.²³ Venous remodeling in the AVF is accelerated by hemodynamic forces in the arterial circulation.²⁴ In the best scenario, the AVF quickly adapts to the new hemodynamic conditions by expanding the intima while favoring outward remodeling. The thickness of the AVF intima varies from mild to extensive, although this characteristic by itself does not determine its stenotic character.^7,21 An eccentric and occlusive neointima may combine with mild medial hypertrophy in early failed fistula.⁹ Conversely, the less eccentric neointima is associated with profound medial hypertrophy in mature AVF. In both cases, myofibroblast-like cells and scattered SMCs populate the intima. It is well accepted that upstream events such as abnormal shear stress,²⁵ anatomical configuration,²⁴ vein diameter,²⁶ and surgical procedures²⁷ determine the extent of IH in the fistula wall. In addition, demographic, etiologic, and comorbid factors that influence AVF outcomes could indirectly modify the degree of vascular remodeling in these vessels.^28,29

The neointimal cell in AVFs

The neointimal cell is one of the most enigmatic entities in vascular biology in terms of its origin, functions, and molecular regulation. The neointimal cell in AVFs is phenotypically similar to those forming culprit lesions in arterial restenosis³⁰ and vein graft vasculopathies.³¹ The cell body contains synthetic and secretory organelles that produce significant amounts of ECM.³² These cells also express a variety of contractile proteins,³³ which suggest the existence of actin bundles that form part of the fibronexus—a specialized adhesion complex that uses transmembrane integrins to allow mechano-transduction from and to the surrounding ECM.³⁴ Whether neointimal cells remain mitotically active in the AVF is uncertain. One study revealed up to 18% of proliferating cells (PCNA⁺) in the intima of human AVFs obtained at the time of surgical revision.³⁵ On the other hand, Hofstra et al.³⁶ described only a very small numbers of mitotic cells (0.9%–3.7%) in similar specimens using Ki-67, a more accurate proliferation marker. Interestingly, Chan et al.³⁷ demonstrated a temporal pattern of cellular proliferation by vascular layer in the swine AVF. Cell proliferation first appears in the adventitia within 2 days after surgery, and later within the intima-media and endothelial line at 7 days. Proliferating cells in all three layers of the wall is almost nonexistent by 42 days postop, suggesting that cell division is primarily an early event.

The contribution of adventitial fibroblasts to the AVF intima

The fibroblast is a quiescent cell without contractile stress fibers (microfilaments) that resides in the adventitia of normal veins.^38,39 The adventitial fibroblast population is highly heterogeneous in terms of structure and function.^40,41 They display a variety of morphology and adhesive structures in vitro. In addition, the adventitial fibroblast responds to changes in wall tension by acquiring a contractile apparatus formed by microfilaments, fibronexus junctions, and other features shared by SMCs.⁴² Activation of fibroblasts can then be detected with specific antibodies for smooth muscle actin (SMA) and vimentin.⁴² The changes in ECM produced by myofibroblasts help maintain hemostasis during the final remodeling and adaptation of the AVF to arterial flow.⁴³ Evidence from in vitro cultures⁴⁴ and animal models for restenosis^45,46 indicates that adventitial myofibroblasts have the extraordinary capacity to migrate toward the intima in response to trauma and contribute in part to neointima formation.

There is a belief that adventitial myofibroblast are the major contributors to neointima formation in the AVF. This notion was initially founded on circumstantial evidence that described myofibroblasts in the adventitia of experimental AVFs as early as 3 days after creation.⁴⁷ The myofibroblasts in the adventitia proliferate earlier than those in the intima³⁷ suggesting that activation of these cells is required for remodeling. Additional studies support the mobilization of adventitial myofibroblasts toward the intima during remodeling of the vein. Using a porcine model of interpositional vein grafts, Shi et al.⁴⁸ showed localization of BrdU-labeled adventitial myofibroblasts from the recipient’s blood vessels in the subendothelial space of the graft. More recently, Misra et al.⁴⁹ tracked the temporal appearance of BrdU⁺ adventitial cells (proliferating) in the venous segment of a porcine arteriovenous graft (AV) graft. These proliferating cells were found at the adventitia-media junction early after anastomosis and then moved toward the intima as the vessel remodeled. Of note, these studies are not conclusive for AVF as profound differences exist between the biological mechanisms underlying the adaptation of both access to the arterial circulation. Therefore, the relative contribution of adventitial fibroblasts to intimal thickening of the AVF remains an open question.

The contribution of local SMCs to the AVF intima

One of the most striking characteristics of SMCs is their extraordinary plasticity to undergo phenotypic modulation/switching from a “contractile” to a “synthetic” phenotype.⁵⁰ Indeed, it is hard to differentiate a “synthetic” SMC from a myofibroblast.⁵¹ At present, the presence of smoothelin is the most reliable marker to distinguish between the differentiated myofibroblasts and “synthetic” SMCs.⁵² The plasticity of SMCs was described early in the 70s, when pioneer studies noted that human SMCs did not retain their morphological and functional characteristics for extended generations in vitro.⁵¹ These “synthetic” SMCs lose contractility features while gaining the capacity to synthesize significant amounts of ECM.⁵¹ As a result, these cells present a larger rough endoplasmic reticulum and numerous free ribosomes⁵³ dedicated to ECM production. They also show an unusual capacity to proliferate in response to growth factors.⁵⁴ Cells with this phenotype express low levels of the contractile SMC markers.⁵⁰ New developments have clearly established that SMC plasticity goes beyond two simple cellular phenotypic stages and that SMCs may acquire more complex phenotypes.⁵⁵ In fact, in vitro the SMC phenotype is influenced by the species, method of isolation, culture conditions, pre-existing disease conditions, and donor’s age.^55,56 The molecular mechanisms dictating SMC plasticity is out of the scope of this paper; however, we recommend review articles by Dr. Gary Owens and collaborators for a better understanding of this process.^44,50,57,58

It is almost certain that SMCs contribute to the neointimal cell population in AVFs. Circumstantial evidence supporting this hypothesis consists of longitudinal SMCs within the AVF neointima staining positively for SM-MHC, desmin, and SMA, the classic markers for differentiated and “contractile” SMCs.^9,59 The presence of these cells in the intima suggests the migration of medial cells into the subendothelial space during remodeling of the AVF. However, more experimental lines of evidence are required to ascertain whether these cells are truly SMCs and not myofibroblasts that acquired the classic “contractile” SMC markers under certain circumstances like in the case of Dupuytren’s disease.⁶⁰ A significant amount of experimental data does support the contribution of venous and arterial SMC to the AVF intima. We recently demonstrated that neointimal cells originate from pre-existing cells in the vein using a chimeric AVF construct that combined wild type (WT) and GFP-labeled veins from transgenic rats. As expected, GFP⁺ neointimal cells were found only in the GFP segment of the fistula, with minimal or no contribution of cells from the adjacent WT segment.⁶¹ Zhao et al.⁶² later confirmed that only GFP⁺ SMCs populated the media and intima of AVFs created in lineage reporter mice (Myh11-Cre/ERT2-mTmG) that specifically expressed the fluorescent protein in SMCs after tamoxifen injection. The contribution of SMCs from the arterial anastomosis to the fistula intima has been suggested by Dr. Jizhong Cheng’s team. These authors found abundant GFP⁺ neointimal cells in AVFs created in a mouse that expressed GFP on arterial SMCs (Wnt1-cre-GFP mice) but not in the vein.⁶³ They further suggested that migration of these cells was partially dependent on circulating FSP-1 expressing fibroblasts that infiltrate the venous wall after anastomosis through Notch dependent mechanism.⁶⁴ These studies failed to rule out the possibility of ectopic activation of these promoters in venous cells during arterialization of the fistula.

The contribution of circulating bone marrow (BM) progenitor cells to the AVF intima

The long-standing paradigm that neointimal cells originate from pre-existing cells in the vascular wall has been recently challenged by studies proposing that BM-derived progenitor cells contribute to intimal expansion in veins and arteries. Indeed, the controversy was sparked when Sata et al.⁶⁵ injured the femoral artery of chimeric mice carrying ROSA26 BM cells that constitutively express the bacterial β-galactosidase (LacZ) gene. Four weeks after injury, they found a significant proportion of neointimal (63.0%) and medial cells (45.9%) staining positively for both LacZ and SMA, a marker for SMC/myofibroblasts. This was interpreted as a strong evidence for the contribution of BM-derived cells to the intimal cell population. Since then, numerous reports have shown a wide diversity of results in the homing and incorporation of BM-derived cells to the intima of mechanically injured arteries⁶⁶ and autologous vein grafts.⁶⁷ However, the multiple methodological limitations frequently observed in these studies have significantly dimmed the enthusiasm for this novel hypothesis.^68-70 The most relevant limitations in these studies include: (1) detection of labeled cells in unfixed tissues where the tracer marker could potentially diffuse from sectioned cells, (2) lack of resolution to discriminate GFP⁺ or LacZ⁺ cells from arterial background (tissue autofluorescence or senescence-associated beta-galactosidase activity), (3) lack of high-resolution confocal microscopy to show definitive co-localization of BM and SMC lineage markers in single cells, (4) lack of appropriate experimental controls to detect cell fusion events, (5) presence of confounding factors (e.g. irradiation) that may modify normal vascular repair process, (6) failure to provide compelling evidence regarding expression of definitive markers of SMC lineage such as SM-MHC, and (7) severe damage of the vessel including complete loss of endothelium and extensive medial SMC apoptosis. More recently, several seminal studies from our group and others that overcome the above described limitations have shown that indeed the contribution of BM-derived cells to the post-injury arterial neointima is minimal or none.^71,72

In the case of AVFs, the contribution of circulating progenitor cells to neointima formation has been directly assessed in three independent studies. In 2006, Castier et al.⁷³ created AVFs in C57/BL6J chimeric mice that had received BM cells from SM-LacZ transgenic mice after irradiation. The SM-LacZ mouse model expresses the reporter gene only in SMCs or SMC-like cells. Surprisingly, the murine AVF intima showed no LacZ staining indicating that neointimal cells in those experimental AVFs did not come from the BM. Three years later, Kokubo et al.⁷⁴ extended the previous results to a model where AVFs were created in chimeric mice that had undergone renal ablation to cause kidney failure. Three weeks after the AVF creation, WT mice reconstituted with GFP⁺ BM had minimal fluorescence in prominent IH lesions, confirming that the BM does not contribute significantly to lesion formation in experimental AVFs. Our group has also investigated the contribution of circulating BM cells to the neointima in a rat model of AVF stenosis.⁶¹ We created fistulas in chimeric rats carrying GFP⁺ BM cells. In agreement with previous studies, we found that GFP⁺ SMA⁺ cells (neointimal cells) in the AVF wall were extremely rare. In summary, unlike all the controversy about the contribution of BM cells to the intima of injured arteries, it seems clear that BM circulating progenitor cells rarely acquire a myofibroblastic phenotype in the fistula wall to contribute to the remodeling of the intima.

The potential contribution of ECs to the AVF intima

New developments have revealed that the local sources of neointimal cells could be more diverse than those traditionally considered, that is, resident SMCs and adventitial fibroblasts. The possibility that ECs have the capability to transition to a mesenchymal or SM-like phenotype in a process known as endothelial to mesenchymal transition (EnMT) has been considered. In vitro, ECs derived from the adult bovine aorta convert to spindle-shaped SMA-expressing cells when treated with TGF-β.⁷⁵ Certain murine endothelial-like cell lines also irreversibly transform into mesenchymal cells upon overgrowth in culture.⁷⁶ One concern raised regarding early reports describing EnMT was the possibility that the primary EC cultures were simply contaminated with small numbers of mesenchymal cells. Recent studies have indicated that EnMT could contribute to the progression of diabetic nephropathy, diabetic renal fibrosis, and cardiac fibrosis.⁷⁷ However, so far, no study has demonstrated that EnMT contributes to neointima formation in AVF. While EnMT could potentially contribute to the neointima in secondary failure, its role in early failure is unlikely as most of the venous ECs are severely damaged by the extreme shear stress produced by the arterial circulation after anastomosis.

The potential contribution of vascular (local) progenitor cells to the AVF intima

Alternative ideas have recently emerged suggesting the presence of progenitor cells (also known as “adult somatic stem cells”) within the vascular wall of arteries and veins that can terminally differentiate into SMCs and ECs during remodeling of the vasculature. Tintut et al.⁷⁸ first described these cells in cultures of bovine aortic medial cells almost two decades ago. These authors identified a subpopulation of mesenchymal-like stem cells that have the potential to produce multiple lineages. More lately, Tang et al.⁷⁹ published a provocative article the revealed the existence of multipotent vascular progenitor cells that express the stemness markers Sox17, Sox10, and S100β. These cells were found in both arteries and veins, were clonable, and differentiated into SMCs in models of vascular injury. Furthermore, Hu et al.⁸⁰ localized progenitor cells in the adventitia of the mouse aortic root that are capable of populating the intima of vein grafts in ApoE-deficient mice after perivascular cell transplantation. These Sca-1⁺ cells also displayed a potential to differentiate into adipogenic, osteogenic, or chondrogenic lineages in vitro.⁸⁰ These authors concluded that during pathological conditions in vein grafting, adventitial progenitor cells participate in the development of vascular disease via differentiation into SMCs. Another study demonstrated that healthy mouse arteries host at least 6% of “side-population” cells known by their stemness properties.⁸¹ In humans, a small number of cells expressing stem cells markers (CD34, Sca-1, c-Kit, and VEGFR2) were identified in neointimal lesions and the adventitia with variable expression of labels.^82-84 Thus, there is sufficient evidence that supports the presence of vascular progenitor cells in vessels which can be a source for SMCs, ECs, and macrophage-like lineages during vascular remodeling.

The idea that vascular progenitor cells can contribute to the AVF intima was approached by Caplice et al.⁸⁵ These authors found c-Kit⁺ cells that also stained positively for SMC and EC markers in the adventitia and neointima in experimental fistulas. We have extended these results to a new murine model of AVF stenosis, created by anastomosing the left renal vein to the abdominal aorta after unilateral nephrectomy.^47,86 c-Kit⁺ cells appeared in these fistulas at day 3 postop, reached their maximum count at day 14, and then decreased to a non-significant level at day 30. Interestingly, the rise in the number of c-Kit⁺ cells correlated with increasing intimal thickness. However, additional efforts are required to demonstrate the pluripotency and ability of these c-Kit⁺ cells to contribute to the formation of the AVF intima.

Implications of neointimal cell origin for therapies targeting intimal hyperplasia

The fundamental reason for the perpetual failure of therapies targeting IH is the lack of solid biological criteria for target, delivery, and drug selection. Clinical trials aiming to treat AVF dysfunction have been designed based on mechanisms extrapolated from arterial restenosis that have been barely demonstrated in the setting of AVFs. For instance, delivery of cytostatic drugs ( sirolimus and paclitaxel) to vascular access with perivascular scaffolds or eluding balloons has inconsistent efficacy outcomes across studies and short-lasting therapeutic effects that are overcome by recurring IH after several months.^87,88 These treatments are inherently nonspecific toward the intended cell type, off targeting ECs, and contractile SMCs that are essential for the proper vein’s function and the resolution of inflammation that leads to fibrosis. We have the imperious necessity to develop cell-specific therapies with minimum side effects on other vascular cells. The first step toward the latter would be to finally elucidate the actual contribution of adventitial myofibroblasts, local progenitors, and synthetic SMCs to the pool of neointimal cells in AVFs. This information serves three main purposes in optimizing drug design: (1) to study drug action and efficacy on the intended cell type, (2) to assess the optimal site (endovascular vs perivascular) and method of drug delivery, and (3) to ensure specificity and avoid significant collateral damage to other structural cells. The final demonstration that adventitial fibroblasts play an important role in the development and growth of AVF IH will open the opportunity to repurpose anti-fibroblast therapies effective in the fight against cancer. These therapies target the fibroblast activating protein (FAP) with low molecular weight inhibitors, prodrugs, antibodies, and FAP-CAR T cells, and vaccines.⁸⁹ In addition, it would allow us to target neointimal cell precursors proactively at the time of AVF creation to prevent excessive postoperative IH. This is a titanic, but not an impossible, task that is feasible by adapting new cell-specific targeting technology using nanoparticles and cell-specific antibodies that have been already used successfully at diverting drugs from organs to prevent systemic toxicity.⁹⁰

Conclusions

The myofibroblastic neointimal cell in AVFs may be one of the examples that suits the “one cell, multiple origins” hypothesis. The intent of this review was to present the current state of knowledge regarding the potential anatomical sources contributing to neointima formation during AVF remodeling. We reveal the complexity of the controversy about the origin of these neointimal cells and identify methodological flaws and over-interpretations that have promoted confusion rather than clarity over the years. Additional studies are required for a better understanding of the anatomical source(s) of neointimal cells in AVFs, using relevant models that address the differences between AVF remodeling and arterial restenosis.