Endothelial Cell Global Positioning System (GPS) for Pulmonary Arterial Hypertension – Homing in on Vascular Repair

Pulmonary arterial hypertension (PAH), defined as a mean pulmonary artery pressure exceeding 25 mm Hg in the presence of a normal capillary wedge pressure¹, is an infrequent (reported incidence 1-2 per million), chronic, and eventually fatal disorder characterized by obliterative microvascular changes, endothelial cell (EC) dysfunction and vascular smooth muscle cell (VSMC) overgrowth. The etiology of PAH varies (idiopathic, familial, or secondary to autoimmune phenomena and infections such as human immunodeficiency virus) but overall outcome (median survival 2.8 years) is universally poor².

Current treatment algorithms involve general supportive measures and pharmacotherapy (calcium channel blockers, anticoagulants, diuretics), as well as targeting major pathways that regulate pulmonary vascular tone: endothelin (ET), nitric oxide (NO) and cyclic adenosine monophosphate (cAMP)³. Therapeutic agents such as ET receptor antagonists (Bosentan), cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 inhibitors (Sidenafil), soluble guanylate cyclase (sGC) activators (Riociguat) and prostacyclin analogues that increase cAMP production (Epoprotenol) provide symptomatic benefit and increased functional capacity⁴. However, they fail to substantially improve life expectancy or reverse the underlying disease process. Potentially curative therapies, other than lung transplantation (which itself has significant attendant morbidity secondary to chronic immunosuppression), that directly modulate the underlying pathogenesis of PAH remain elusive.

While there remains much to understand regarding the etiology of PAH, novel therapeutic interventions have been developed to enable mitigation of disease in pre-clinical (animal) models by targeting known activated pathways in PAH. Preventing disease progression by lung microvasculature repair is one such emerging therapeutic intervention⁵. Given the success arising from proof-of-concept animal studies, cellular therapy has become an increasingly attractive avenue for potential clinical application, reaching Phase II and III trials⁶ (NCT01795950). Such strategies have evolved from bone marrow transplantation, and are now the subject of intense interest to regulate the innate and adaptive immune system following solid organ transplantation. This has provided a platform for the utilization of other cell types (including stem and terminallydifferentiated cells) in a variety of disease processes (www.clinicaltrials.gov).

The chemotactic cytokine CXCL8, also known as interleukin (IL)-8, is produced by phagocytic cells to promote neutrophil accumulation at sites of injury. CXCL8 is also produced by EC (in response to hypoxic stress)^{7, 8}, VSMC^{9, 10}, and epithelial cells^{11, 12}. CXCL8 is implicated in lung injury arising from a variety of causes including ischemia reperfusion injury (IRI)¹³, aspiration¹⁴, and sepsis¹⁵. Although not specifically documented in PAH, induction of pro-inflammatory cytokines (including CXCL8) has been associated with increased mortality¹⁶. The two CXCL8 receptors, CXCR1 (IL-8RA) and CXCR2 (IL-8RB), share sequence homology and high affinity for the ligand, and promote via ligand-receptor binding, G protein and phospholipase C activation¹⁷. Downstream effector mechanisms through Ras, Akt and mitogen-associated protein kinase trigger neutrophil adhesion, transmigration and degranulation¹⁸.

In a novel and interesting study by Fu et al.¹⁹ in the current issue, the infusion of EC transfected to express IL8RA/B reduced acute pulmonary inflammatory cell influx and cytokine release to mitigate endothelial damage following monocrotaline (MCT) injury. A single infusion of IL8RA/B-expressing EC resulted in a beneficial effect on the development of MCT-driven PAH with attenuated right ventricular hypertrophy and decreased VSMC overgrowth. The authors also found restoration of alveolar and arteriolar endothelial nitric oxide synthase (eNOS)-positive cells and reduced inflammatory cell (macrophage and neutrophil) infiltrate.

This study, and others from the same group^{20, 21}, utilize terminally-differentiated EC and thus represent a departure from previous investigations employing cells with greater self-renewal capacity (reviewed in ²²). Endothelial progenitor cells (EPC) have been shown to contribute to repair of damaged endothelium through neoangiogenesis^{23, 24}. Unlike other EC populations, EPC do express CXCR2 (IL8RB), which mediates adhesion to both matrix in vitro and recruitment to areas of carotid arterial injury in vivo in a CXCR2-dependent manner²⁵. Pathological changes within damaged organs may also affect cell homing; for example chronic hypoxia may increase peripheral EPC mobilization, but pulmonary recruitment is limited²⁶. This is potentially relevant as the right ventricle, though not a direct target of MCT, nonetheless experiences injury in PAH and may represent another location for transfused cells homing.

One disadvantage of cell-based therapies is the substantial proportion of cells lodging within the pulmonary vasculature following intravenous injection. Hence, only a small percentage of effector cells remain capable of reaching the systemic circulation, although this obstacle is exploited when the target organ is the lung as in the present study. Nonetheless, the use of cells transfected with ‘homing devices’ may facilitate tracking of cells to the site of damage, as in the present situation¹⁹ where MCT-mediated endothelial damage initiates CXCL8 production to putatively attract infused CXCL8-receptor bearing EC.

So, how to proceed from here? Several critical questions should be answered if cell therapies are to become a platform for treatment of disease: (i) precisely where do these cells travel after infusion, (ii) do these cells maintain physiological function and (iii) what is their longevity in vivo? The new work from Fu et al. ¹⁹ suggest several other interesting questions (Figure 1). How are these transfected, terminally-differentiated EC cheating death in vivo, now that they no longer have a tissue scaffold or growth factor support, and then how are they mediating vascular repair? Are these cells themselves providing a reparative mechanism for the endothelial layer, modulating healthy or damaged EC function to initiate repair, or are they instead preventing vascular damage by acting as a dominant-negative (decoy) receptor for released IL-8 to reduce inflammatory cell homing? As with all animal models, the relevance of this type of treatment to human PAH, which typically lacks an inciting acute inflammatory event, remains to be established. Also important for the future pursuit of cell-based therapy is the regard for the overall safety of manipulated, albeit autologous, cells.

Figure 1. IL-8 receptor expressing endothelial cells reduces monocrotaline-mediated pulmonary inflammation and PAH.

Monocrotaline (MCT) leads to endothelial cell (EC) injury, interstitial edema, and production of IL-8 by damaged EC and vascular smooth muscle cells (VSMC). The chemotactic cytokine attracts neutrophils (N) to the site of injury. Green fluorescent protein-positive EC transfected to express IL8RA/B and infused systemically at the time of MCT administration home to the site of injury to reduce inflammation, and participate in endothelial repair. F= fibroblast

In conclusion, the field of PAH has yielded to cellular therapy as a potential future therapeutic opportunity, with novel targets and techniques to recover vascular function.