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. Author manuscript; available in PMC: 2022 Nov 14.
Published in final edited form as: Am J Transplant. 2022 Apr 19;22(7):1754–1759. doi: 10.1111/ajt.17050

Wanted: An Endothelial Cell Targeting Atlas for Nanotherapeutic Delivery in Allograft Organs

Samuel T Liburd Jr 1,2, Audrey A Shi 3, Jordan S Pober 4, Gregory T Tietjen 2,3
PMCID: PMC9651180  NIHMSID: NIHMS1844167  PMID: 35373446

Abstract

Despite the profound shortage of organs available for transplant in the U.S., over 5000 donated organs were declined for use in 2020. Many of these organs were declined due to donor comorbidities or preservation injuries that predispose grafts to rejection and loss. The risks of these poor outcomes can potentially be reduced by pre-transplant application of normothermic machine perfusion (NMP). To date, the clinical use of NMP has focused on extending preservation and improving organ assessment, but the opportunity for ex situ therapeutic delivery may be the most transformative aspect of this technology. In this Personal Viewpoint, we argue that the endothelial cells (ECs) that line the graft vasculature are an accessible, under-exploited, and attractive target for transplant therapeutics delivered during NMP. We further contend that molecularly-targeted nanoparticles (NPs) represent a promising therapeutic vehicle particularly well-suited to NMP. However, to achieve this potential, we need to answer three key questions: 1) What EC sub-populations exist within an organ?; 2) How can these cells be accessed?; and most importantly, 3) How can preferential retention of NPs by the cells of interest be maximized? Here we argue for creating an EC-targeting atlas as a body of knowledge that answers these questions.

1). The Case for Endothelial Cell-Targeted Nanomedicines in Transplant

Graft vascular endothelial cells (ECs) sense and respond to factors produced within both the circulation and parenchyma of a donor graft to mediate homeostatic regulation of critical physiologic factors including blood fluidity, vascular tone, and vessel wall permeability (1). ECs also play an essential role in immune responses, acquiring new functions to regulate the progression and resolution of inflammation (1). A breakdown in any of these EC functions—termed ‘EC dysfunction’—can contribute to the loss of transplanted organs. Moreover, because graft ECs are the first point of contact with the recipient immune system, many forms of post-transplant injury initiate at the EC surface, including ischemia reperfusion injury (IRI) and both acute and chronic rejection (2, 3). IRI-induced dysfunction leads to a loss of permselectivity, resulting in vascular leak. Vascular ECs can also participate in adaptive immunity by presenting antigens to circulating to T cells. Based on EC phenotype or activation state they may also trigger memory or regulatory T cell expression (4). Thus, as a nexus of transplant pathology, graft ECs represent important therapeutic targets. However, there are currently no selective EC-targeted therapies in clinical use for transplant, likely due to the significant challenges associated with achieving EC-specific therapeutic effects.

Targeting any therapy specifically to graft EC requires first addressing the fundamental challenge of drug delivery: avoiding off-target drug accumulation. Normothermic machine perfusion (NMP) provides a unique opportunity to reduce the complexity of this challenge by isolating the graft from the systemic circulation. This allows us to focus on the remaining obstacles, namely: 1) Robust and specific therapeutic retention on the EC subtype of interest; 2) Durability of drug effect post-transplant; and 3) Adaptability to address an array of potential pathologies. Developing a drug delivery tool that possesses all three attributes—specificity, durability, and adaptability—requires a modular system that can be tuned along each axis.

The modular design of polymeric NPs makes them particularly well-suited to the task of drug delivery during NMP (5–8). To achieve carrier specificity, the surface, size, and charge of polymeric NPs can be adjusted to minimize cellular interaction with off-target cells. A variety of targeting ligands (e.g. antibodies, peptides) can be attached to the NP surface to enable retention by the EC subtype of interest (7–10). Durability of drug effect can be achieved by manipulating the polymer composition and molecular weight to slow the rate of particle degradation; prolonged drug release spanning weeks to months has been routinely demonstrated (11). Upon internalization, NP degradation often occurs within lysosomal compartments which may vary with EC subtype (12). Finally, the core of polymeric NPs can be adapted to accommodate different therapeutics from small molecules to nucleic acids (11). While these attributes make polymeric NPs an ideal therapeutic delivery vehicle, there is one final hurdle to overcome. We need a roadmap to ensure NPs can achieve close contact with and be locally retained by EC sub-populations across different organs. We refer to this broad collection of knowledge as an ‘EC-targeting atlas.

2). Creating an EC-Targeting Atlas: Building the Roadmap

The first step in creating an EC-targeting atlas is to define ‘roadmaps’ of the organs themselves, i.e. routes to ECs sub-populations of interest (neighborhoods). These roadmaps illustrate how ECs are connected through vessels with unidirectional flow in both native and NMP settings (Figure 1). In the sections below, we highlight challenges to navigating the local neighborhoods (i.e. EC sub-populations) in the four most transplanted solid organs: kidney, heart, lung, and liver. Arterial and venous ECs are important, but they are also well-recognized by widely shared EC surface molecules and generally well-accessed by flow during NMP (13). Therefore, we focus primarily on microvascular and other specialized EC sub-populations (referred to as ‘ECs of interest’) that influence various transplant pathologies (Figure 1) (13).

Figure 1: Building the Roadmaps.

Figure 1:

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Natural routes of flow to desired EC neighborhoods in the kidney (A), heart (B), lung (C), and liver (D) and their changes during NMP. EC: Endothelial cell, GE: Glomerular Endothelium, PTCs: Peritubular Capillaries, RA: right atrium, RV: right ventricle, LA: left atrium, LV: left ventricle LSECs: Liver Sinusoidal Endothelial Cell

2.1). Map of the Kidney:

The microvascular ECs of interest in the kidney are primarily contained within the glomerular and peritubular capillaries. Glomerular ECs (GECs) are supported by both pericyte-like mesangial cells and epithelial podocytes (2, 13). GECs form 60-80 nm pores (fenestrae) without diaphragms and overlie a basement membrane that has a strong positive charge (14). The peritubular capillaries (PTCs) are lined by specialized ECs containing diaphragmed fenestrae which facilitate salt and water exchange with different segments of the tubules (2). In the medulla, ECs in the descending vasa recta—an extension of the PTCs—have a continuous basement membrane and do not form fenestrae. In contrast, ECs in the ascending vasa recta do contain diaphragmed fenestrae, with a higher number in the inner medulla relative to the outer medulla (13). Injuries to GECs or ECs in PTCs have overlapping but distinct contributions to different disease states in the transplanted kidney, such as recurrent focal glomerulosclerosis or IRI, respectively (2). Thus, these EC sub-populations represent potential ‘neighborhoods’ which we might target during NMP. We next consider how to navigate routes of flow to access these neighborhoods during NMP.

Renal NMP recapitulates natural circulatory flow in kidneys. Blood enters through renal arteries, which arborize in the renal cortex to give rise to afferent arterioles. These feed the capillary tufts of the glomeruli that drain through efferent arterioles. The latter give rise to PTCs which nourish the proximal portions of the renal tubules. These vessels then extend into the medulla—referred to as the ‘vasa recta’ (VR) as noted above—to supply the loops of Henley before returning to the cortex to feed the distal portions of the tubules and draining into renal veins (Figure 1A). Because NMP is typically performed anterograde through the renal artery, the charge of the GEC basement membrane and the presence of fenestrae can adsorb and potentially deplete a large fraction of NPs prior to reaching the PTCs and VR. Larger, negatively-charged polymeric NPs (over ~50 nm in diameter) can potentially be used to reduce this loss. Additionally, NMP of the kidney can theoretically be performed retrograde through the renal vein, providing direct access to the VR and PTCs. Employing a combination of these approaches may optimize access to any renal microvascular EC sub-populations.

2.2). Map of the Heart:

ECs of interest in the heart include the microvascular ECs of the coronary vessels and those that line the heart chambers (termed ‘endocardium’) and valves. In the coronary circulation, ECs overlie a continuous basement membrane (13). In response to prolonged ischemia, these coronary capillaries can experience occlusion and thus resist perfusion (‘no reflow’), making coronary capillary ECs important therapeutic targets (15). The endocardium and valves also serve important functions in maintaining homeostasis and therefore may necessitate treatment during NMP. ECs in these regions contain numerous microvilli and gap junctions that increase permeability, surface area, and blood regulatory capacity (13).

In the heart, significant differences between natural flow and NMP limit access to certain EC sub-populations. Natural perfusion of the heart starts with systemic venous return to the right-side heart and exits through the pulmonary arteries to the lungs. After oxygenation, blood returns to the left heart through the pulmonary veins and exits the left heart through the aorta. Coronary arteries arise from the proximal portions of the ascending aorta, run along the epicardial surface of the heart, and give rise to penetrating arteries. These arteries branch within the myocardium into arterioles that drain into coronary veins and empty through the coronary sinus into the right atrium (Figure 1B). Typical NMP of the heart differs from this natural circuit in that perfusate enters through the aorta, as opposed to the right atrium (16). The aortic valve is closed to ensure antegrade coronary perfusion. Any perfusate that enters the left ventricle is then vented out of the organ (16). While coronary ECs may be easily accessible, ECs of the left-heart endocardium or valves may be more challenging to reach during NMP. This means we may need to either create alternative routes or change the design of NMP to access these EC neighborhoods.

2.3). Map of the Lung:

In the lungs, ECs of interest include both alveolar and bronchial capillary ECs. Alveolar capillaries contain a specialized type of ECs, known as ‘aerocytes’, which are larger cells with numerous “Swiss-cheese-like” pores and a thin basement membrane to facilitate gas exchange (17, 18). Also present alongside aerocytes are more typical capillary ECs that maintain homeostasis and uniquely function to help repair the lung (17). Bronchial ECs, which line the vessels that supply the conducting airway epithelium, represent another important EC sub-population. The loss of the bronchial microcirculation post-transplant may lead to increased hypoxia in the airway epithelium and increase the risk of patients developing bronchiolitis obliterans (19).

As in heart, there are also critical differences between natural flow and flow during NMP in the lung. In vivo, the lung is supplied by both the pulmonary and bronchial circulation. The pulmonary circulation starts with the pulmonary artery, arising from the right heart before branching into the alveolar capillaries in the lung, and returning to the left heart through the pulmonary veins. The bronchial circulation starts from the aorta or intercostal arteries and branches into capillaries that supply the epithelial cells of the lung before converging into the bronchial veins (13, 19) (Figure 1C). These veins drain into either the pulmonary veins in the lungs or the IVC at the lung hilum (13). Because the bronchial venous drainage does not converge to one vessel and the bronchial artery is not preserved when the transplanted lung is removed, current reperfusion of the lung only restores the pulmonary circuit (19). As such, while alveolar ECs are readily accessible during NMP, treatment of bronchial ECs may pose more of a problem.

2.4). Map of the Liver:

The liver contains two broad EC classes of interest: liver sinusoidal endothelial cells (LSECs) and ECs of the peri-biliary plexus. The sinusoids are lined with LSECs that form large (150-175 nm in diameter) un-diaphragmed fenestrae and rest upon a discontinuous basement membrane. These anatomic features allow free fluid and nutrient exchange between blood and hepatocytes (13, 20). Upon injury, sinusoids may be converted into continuous capillaries (‘capillarization’) in which LSECs are replaced by conventional continuous EC lining, limiting access of blood-borne substances to the hepatocytes (20). Unlike the complex ECs of the sinusoids, the ECs of the peri-biliary plexus form capillary extensions of the hepatic artery which has a continuous basement membrane (21).

The liver contains two perfusion circuits: the hepatic circulation arising from the celiac artery, and the portal vein which drains blood from the gut. However, instead of converging at the venules (as in the lung), the hepatic and portal systems converge in the portal triads which drain into the liver sinusoids. These empty into central veins that drain through a common hepatic vein into the inferior vena cava. The hepatic artery supplies both the LSECs and independent micro-vessels of the peri-biliary plexus. Liver NMP typically includes perfusion of both the hepatic and portal circulation. Perfusion of the liver through the hepatic artery can provide direct NP access to only the peri-biliary ECs on first pass, however, the same is not true of LSECs. LSECs are in close proximity to Kupffer cells (i.e. resident macrophages within sinusoids) (13, 20). Because Kupffer cells are highly phagocytic, their location and abundance within the liver sinusoids can further complicate LSEC-specific targeting. Thus, accessing LSECs while avoiding uptake by Kupffer cells may constitute the most challenging prospect for EC-specific delivery during NMP.

3). Creating an EC-Targeting Atlas: Populating the Roadmaps with Landmarks

Above, we highlighted the roadmaps of our targeting atlas, identifying some important EC sub-populations and routes of access in each organ. In addition to this framework, we must ensure NP retention and internalization by the appropriate EC-sub-populations to take full advantage of sustained local drug delivery. Thus, a key task in developing an EC-targeting atlas is to identify target receptors on EC sub-populations that support specific retention of targeted NPs. Some specific receptors in human ECs have been identified (e.g. CD32 in the LSECs and PV-1 in the kidney PTCs), but it as yet unclear if these can support robust NP targeting (14, 20). Moreover, even with an exceptional target, we are still limited if we do not have an effective targeting ligand. Here, we define these optimal combinations of ligand/receptor pairs as landmarks in the context of our EC-targeting atlas and further suggest methods to define optimal landmarks for each EC population.

There are two general approaches that can be used to identify optimal landmarks for our EC targeting atlas. The first—and more established—approach uses combinatorial libraries of binding molecules (‘binders’) to first identify binders to the ECs of interest with a method commonly referred to as ‘biopanning’; we refer to this as the ‘binder-first’ approach. An alternative ‘receptor-first’ approach focuses on first defining which receptors are most abundantly expressed and accessible on the luminal surface of the EC of interest, before then generating the binders against these receptors. In the following sections, we describe these two approaches in more detail and discuss some of the considerations in applying either approach during NMP.

3.1). The Binder-First Approach:

A binder-first approach focuses on allowing the EC subtype to dictate selection of a specific binder without advanced knowledge of the receptor. An archetypal example used phage libraries with randomly generated peptides delivered either in vivo or ex vivo in a process known as ‘biopanning’ (22). In one study, adherent phages were collected, expanded, and rescreened repetitively on isolated membranes, enriching in each cycle for phage with the desired specificity. These phage were then introduced in vivo in rats to demonstrate organ-specific targeting (23). Despite the efficacy of this approach in animal models, there is no guarantee the same binders will work in humans (23–25). Nevertheless, the same approach could be adapted for isolated human organs and/or ECs.

In human organs undergoing NMP, a binder-discovery approach has four main steps: isolation, biopanning, binder selection, and target validation (Figure 2). Isolation can refer to either the entire organ on NMP or in vitro suspensions of EC cells/membranes for binder selection. Next, biopanning occurs via a combinatorial library used for a cyclic process of positive and negative selection to enrich specific binders (positive) and remove non-specific binders (negative) until a few optimal binders have been selected. These binders can then be used to pull down the cognate receptor for identification by mass spectrometry (23).

Figure 2: Populating the Roadmaps.

Figure 2:

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To identify landmarks one can use either binder-first or receptor-first discovery approaches. Binder-First discovery: In this approach, human tissue is isolated and then screened with a combinatorial library—(‘biopanning’) to select for binders to ECs of interest. Immunoprecipitation and mass spectrometry can identify bound receptors of interest. Receptor-first discovery: In this approach, isolated whole organs are dissociated and sorted using FACS (Fluorescence-activated cell sorting). Single cell transcriptomics can identify EC subpopulations and receptors that localize to the cell membrane. A variety of imaging or mass spectrometry techniques may be used to characterize receptor abundance. For both approaches binders must be identified or generated, the spatial location confirmed in tissue samples, and targeting efficacy measured during NMP. EC: Endothelial Cell, NP: Nanoparticle

3.2). The Receptor-First Approach:

The receptor-first approach also has four main steps: isolation, sorting, receptor identification and characterization, and target validation (Figure 2). Isolation can begin with generating single cell suspensions from declined human organ tissues that can then be sorted into EC subpopulations using fluorescence-activated cell sorting (FACS). Single cell transcriptomic analysis of these cells can be used to retain EC-subpopulations of interest using markers such as CD31, or remove lymphatic ECs with Lyve-1 or Prox-1 (26). Furthermore, novel subclassifications of organ-specific ECs are being rapidly discovered (17). However, while these markers are useful to identify different EC subgroups, they may not make ideal targets. Further analyses of single cell transcriptomic data are necessary to identify accessible landmarks of high abundance on the luminal surface. Candidate receptors can be confirmed at the protein level using higher throughput imaging techniques like cyclic immunofluorescence or mass spec imaging (27–29). Single cell mass spec may also become a viable validation approach as the technology matures (30). Once a candidate receptor is identified, an appropriate targeting molecule can be either be selected from commercially available options or generated through protein engineering (e.g. phage display).

3.3). Testing the Atlas – Targeting Validation During NMP:

The benchmark of any good atlas is its ability to reliably direct a user to a location of interest. Likewise, the landmarks identified by our targeting atlas needs to be validated during NMP. Here, we can leverage the unique potential of NMP to serve as a setting for pre-clinical validation using transplant declined human organs. NPs that have been conjugated to the developed targeting molecules can be loaded with fluorescent dye, small molecule drugs, or RNAi and introduced during perfusion to confirm the efficacy of the targeted NPs in a setting with directly translational relevance. Because of the variability inherent in human organs, these validation experiments are necessary to confirm how consistently a landmark can be used to target EC sub-populations in different donors. Thus, as we collect more data, the information within our EC-targeting atlas can be enriched to describe how landmarks may differ between donors and in the context of different pathologies.

4). Beyond NMP: The utility of an EC-targeting atlas for in vivo drug delivery

Creating an EC-targeting atlas for therapeutic delivery generates opportunities for transplant beyond NMP. For example, Nadig and colleagues have described immunosuppressive efficacy of targeted NPs in other transplant settings such as cold storage (9). Thus, in combination with an EC-targeting atlas, new and improved targeting ligands for a variety of NP applications may be developed (9, 10). This utility may also extend to in situ therapies for end-stage organ failure. After all, ex vivo drug delivery in isolated organs can be seen as a model of intra-arterial drug infusion in vivo via interventional radiology. Using this atlas to better target drugs can help us circumvent issues of extreme toxicity associated with many current in vivo therapies. Thus, an effective EC targeting atlas may hasten a new class of precision transplant therapies.

Abbreviations:

EC

endothelial cell

GEC

Glomerular endothelial cell

IRI

Ischemia reperfusion injury

LSECs

Liver sinusoidal endothelial cells

NMP

normothermic machine perfusion

NP

nanoparticle

PTC

peritubular capillary

VR

Vasa recta

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