Yon K. Sung

Regulatory T Cells Limit Vascular Endothelial Injury and Prevent Pulmonary Hypertension

Rationale: Pulmonary arterial hypertension (PAH) is an incurable disease associated with viral infections and connective tissue diseases. The relationship between inflammation and disease pathogenesis in these disorders remains poorly understood. Objective: To determine whether immune dysregulation due to absent T-cell populations directly contributes to the development of PAH. Methods and Results: Vascular endothelial growth factor receptor 2 (VEGFR2) blockade induced significant pulmonary endothelial apoptosis in T-cell-deficient rats but not in immune-reconstituted (IR) rats. T cell–lymphopenia in association with VEGFR2 blockade resulted in periarteriolar inflammation with macrophages, and B cells even prior to vascular remodeling and elevated pulmonary pressures. IR prevented early inflammation and attenuated PAH development. IR with either CD8 T cells alone or with CD4-depleted spleen cells was ineffective in preventing PAH, whereas CD4-depleting immunocompetent euthymic animals increased PAH susceptibility. IR with either CD4+CD25hi or CD4+CD25− T cell subsets prior to vascular injury attenuated the development of PAH. IR limited perivascular inflammation and endothelial apoptosis in rat lungs in association with increased FoxP3+, IL-10- and TGF-&bgr;-expressing CD4 cells, and upregulation of pulmonary bone morphogenetic protein receptor type 2 (BMPR2)–expressing cells, a receptor that activates endothelial cell survival pathways. Conclusions: PAH may arise when regulatory T-cell (Treg) activity fails to control endothelial injury. These studies suggest that regulatory T cells normally function to limit vascular injury and may protect against the development of PAH.

Blocking Macrophage Leukotriene B4 Prevents Endothelial Injury and Reverses Pulmonary Hypertension

In a rat model of pulmonary hypertension, inhibition of LTB4 synthesis in macrophages that accumulate in lung tissue reverses the disease. How to Open a Blocked Vessel Like the pressure that builds up in a kinked garden hose, pulmonary hypertension occurs when the blood vessels in the lung become occluded. This hard-to-treat disease can arise in various settings, sometimes along with collagen vascular disease or HIV infection. It ultimately leads to heart failure as the heart tries to pump against higher resistance. Now, Tian and her colleagues show that certain types of pulmonary hypertension may be caused by a leukotriene B4 (LTB4) released from the macrophages that accumulate in lung tissue and that interruption of this process can reverse the disease. Although much of their evidence comes from a rat model of hypertension, the same may be true of some patients as well. Treatment of athymic rats with the tyrosine kinase inhibitor SU5416 causes them to acquire pulmonary hypertension. At the same time, macrophages gather around the small arterioles of the lung and synthesize an excess amount of LTB4. This leukotriene injures the endothelial cells of the nearby vessels, causing apoptosis while simultaneously provoking abnormal proliferation of the smooth muscle cells. This excess cell division results in arterial occlusion and hypertension. The authors found that damping down excess LTB4 by inhibiting its biosynthesis could reverse disease: In treated animals, cardiac function improved and obstructed arterioles opened. These results may apply to certain patients with pulmonary hypertension: Among a group of 19 patients, those that had pulmonary hypertension secondary to a connective tissue disease generally show higher LTB4 in serum. The next step will be to see whether therapies directed toward the LTB4 signaling system can help to clear the arterioles in patients with pulmonary hypertension, at least in those with associated inflammation. Pulmonary hypertension (PH) is a serious condition that affects mainly young and middle-aged women, and its etiology is poorly understood. A prominent pathological feature of PH is accumulation of macrophages near the arterioles of the lung. In both clinical tissue and the SU5416 (SU)/athymic rat model of severe PH, we found that the accumulated macrophages expressed high levels of leukotriene A4 hydrolase (LTA4H), the biosynthetic enzyme for leukotriene B4 (LTB4). Moreover, macrophage-derived LTB4 directly induced apoptosis in pulmonary artery endothelial cells (PAECs). Further, LTB4 induced proliferation and hypertrophy of human pulmonary artery smooth muscle cells. We found that LTB4 acted through its receptor, BLT1, to induce PAEC apoptosis by inhibiting the protective endothelial sphingosine kinase 1 (Sphk1)–endothelial nitric oxide synthase (eNOS) pathway. Blocking LTA4H decreased in vivo LTB4 levels, prevented PAEC apoptosis, restored Sphk1-eNOS signaling, and reversed fulminant PH in the SU/athymic rat model of PH. Antagonizing BLT1 similarly reversed established PH. Inhibition of LTB4 biosynthesis or signal transduction in SU-treated athymic rats with established disease also improved cardiac function and reopened obstructed arterioles; this approach was also effective in the monocrotaline model of severe PH. Human plexiform lesions, one hallmark of PH, showed increased numbers of macrophages, which expressed LTA4H, and patients with connective tissue disease–associated pulmonary arterial hypertension exhibited significantly higher LTB4 concentrations in the systemic circulation than did healthy subjects. These results uncover a possible role for macrophage-derived LTB4 in PH pathogenesis and identify a pathway that may be amenable to therapeutic targeting.

A brief overview of mouse models of pulmonary arterial hypertension: problems and prospects

Many chronic pulmonary diseases are associated with pulmonary hypertension (PH) and pulmonary vascular remodeling, which is a term that continues to be used to describe a wide spectrum of vascular abnormalities. Pulmonary vascular structural changes frequently increase pulmonary vascular resistance, causing PH and right heart failure. Although rat models had been standard models of PH research, in more recent years the availability of genetically engineered mice has made this species attractive for many investigators. Here we review a large amount of data derived from experimental PH reports published since 1996. These studies using wild-type and genetically designed mice illustrate the challenges and opportunities provided by these models. Hemodynamic measurements are difficult to obtain in mice, and right heart failure has not been investigated in mice. Anatomical, cellular, and genetic differences distinguish mice and rats, and pharmacogenomics may explain the degree of PH and the particular mode of pulmonary vascular adaptation and also the response of the right ventricle.

Low-Dose FK506 (Tacrolimus) in End-Stage Pulmonary Arterial Hypertension

To the Editor: Despite recent advances in therapy, pulmonary arterial hypertension (PAH), characterized by occlusive vasculopathy of the pulmonary arteries, remains a progressive disease without a cure (1–3). Although currently approved PAH medications have not demonstrated antiremodeling properties in humans, novel antiproliferative strategies have shown some benefits and also raised safety concerns (4–6); for example, none target a genetic predisposition of PAH: dysfunctional bone morphogenetic protein receptor 2 (BMPR2) signaling. Loss-of-function mutations in BMPR2 in patients with familial and idiopathic PAH (7–9) are associated with increased pulmonary vasculopathy (10). Furthermore, reduced BMPR2 expression is observed even in patients without a mutation, reinforcing the importance of decreased BMPR2 in PAH (11). In a high-throughput screen of 3,600 drugs approved by the U.S. Food and Drug Administration, we identified low-dose FK506 (tacrolimus) as a potent BMPR2 activator that reversed experimental PAH (12). We therefore hypothesized that low-dose FK506 would be beneficial in patients with PAH by increasing BMPR2 signaling. On the basis of these findings, we initiated a randomized, double-blind, placebo-controlled phase IIa trial (FK506 [Tacrolimus] in Pulmonary Arterial Hypertension [TransformPAH]) to evaluate the safety and tolerability of FK506 in stable patients with PAH. Here, we report our clinical experience with compassionate use of low-dose FK506 in three patients with end-stage PAH who did not qualify for our trial because of the severity of their illness (patient details are provided in the online supplement). We assessed traditional clinical parameters, New York Heart Association (NYHA) functional class, 6-minute-walk distance, serologic biomarkers, and hospital admissions, as well as protocolized cardiac magnetic resonance imaging assessed by blinded readers (13, 14). All patients continued receiving stable doses of PAH medication and diuretics throughout the 12-month period. The TransformPAH clinical trial was registered with www.clinicaltrials.gov (NCT 01647945). Patient 1 Patient 1 is a 36-year-old historically athletic woman, NYHA-IV, with rapidly progressive idiopathic PAH requiring rapid up-titration of epoprostenol and the addition of sildenafil and ambrisentan for recurrent hospitalizations for right ventricular (RV) failure (Figure 1). Despite aggressive treatment, she still reported NYHA-III/IV symptoms, an elevated N-terminal-pro-B type natriuretic peptide, and a Registry to Evaluate Early and Long-Term PAH Disease Management (REVEAL) risk score of 11, stratifying her as high risk with a potential 1-year mortality risk of 15–30% (3, 15). She was listed for lung transplantation. At that time, she was offered compassionate treatment with FK506 (goal trough blood level, 1.5–2.5 ng/ml). Figure 1. Timeline of symptoms, clinical parameters, events, and therapies for patients 1–3 before and after initiation of FK506. Registry to Evaluate Early and Long-Term PAH Disease Management (REVEAL) risk score, %1-year survival: score 1–7 = 95–100% ... Within 1 month of FK506 initiation, she reported substantial improvement in symptoms (Figure 2). Within 2 months, she was placed on hold for transplantation by the lung transplant team. After 3 months, her 6-minute-walk distance improved by 100 m, her N-terminal-pro-B type natriuretic peptide decreased more than 50%, and she reported NYHA-I symptoms (Figure 1; see Figure E1A in the online supplement). During the 12-month period, cardiac magnetic resonance imaging showed a stable RV ejection fraction, RV end-diastolic volume index, and cardiac index (Figure E1B). Her REVEAL risk score decreased to 3 (range, 3–6), placing her in the low-risk category (Figure 1). Although the 12 months before FK506 were characterized by three hospitalizations for RV failure, the subsequent 12 months were free of any PAH-associated hospitalizations (Figure 1). At the time of this submission, the patient is 27 months from the initiation of FK506, continues to report NYHA-II symptoms, and has been free from hospitalization or clinical deterioration. Figure 2. Biomarkers for patients 1–3 before and after initiation of FK506. (A) Bone morphogenetic protein receptor 2 (BMPR2) messenger RNA (mRNA)/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in peripheral blood mononuclear cells (PBMCs) of healthy ... Patient 2 Patient 2 is a 50-year-old woman with end-stage systemic sclerosis–associated PAH receiving intravenous treprostinil, sildenafil, and ambrisentan, as well as an intravenous dopamine infusion for end-stage RV failure and hypotension. The patient continued to report NYHA-III/IV symptoms and had an elevated N-terminal-pro-B type natriuretic peptide (range, 4,926–15,161 pg/ml) and four hospitalizations for progressive RV failure and palliative paracenteses (Figure 1). Given the lack of further therapeutic options, she was offered FK506. Cardiac magnetic resonance imaging at baseline and 3 and 6 months showed substantial improvement in RV ejection fraction, stable RV end-diastolic volume index, and improvement in right ventricular stroke volume index and cardiac index (Figure E1B), with a reduction back to baseline at 12 months. Her REVEAL risk score decreased from 12 to 11. At 12-month follow-up, she had stable NYHA-III symptoms, a 94-m increase in 6-minute-walk distance, 30% reduction in her N-terminal-pro-B type natriuretic peptide, and no PAH-related hospitalizations since being on FK506 (Figure 1). The patient is currently 26 months post-FK506 initiation and has not experienced further clinical deterioration. Patient 3 Patient 3 is a 55-year-old woman with severe end-stage drugs-and-toxins–associated PAH, NYHA-III/IV, who is receiving high-dose intravenous treprostinil and sildenafil, has an intolerance to endothelin receptor antagonists, is listed for lung transplantation, and was offered FK506. Despite initial symptomatic improvement (Figure 1), the patient voluntarily discontinued FK506 after 4.5 months. Unfortunately, during the ensuing 7 months, she showed progressive clinical worsening, culminating in an intensive care unit admission for RV failure and large pericardial effusion. On the patient’s wish, she was restarted on FK506 and is currently 12 months after her second FK506 initiation, feeling much better, with compensated NYHA-II symptoms and without any further hospital admission for RV failure. Serologic Biomarkers None of the three patients had mutations in BMPR2, SMAD9, or caveolin-1. We measured BMPR2 expression and specific BMPR2-associated genes and molecules (Id1 [inhibitor of differentiation 1], Smurf-1 [SMAD-specific E3 ubiquitin protein ligase 1], IL-6, LIMK1 [LIM domain kinase 1], Cofilin-1, and microRNAs 21 and 27a) at baseline and 3, 6, and 12 months into FK506 treatment in patients versus healthy controls (n = 12) (see online supplement). Patients had significantly lower BMPR2 messenger RNA expression at baseline (Figure 2), with near normalization of BMPR2 and associated genes after 12 months of FK506 treatment. Strikingly, patient 3, who stopped FK506 after 4.5 months and who worsened clinically during the following 7 months, showed a 12-month BMPR2 profile that was opposite that of patients still receiving FK506 therapy. Discussion Our results suggest a potential clinical benefit of low-dose FK506 in end-stage PAH, judged by the patients’ marked clinical response, stabilization in cardiac function, and freedom from hospitalization for RV failure. Despite the overall positive experience, we caution that these findings are highly preliminary. The efficacy of this therapy must be validated in appropriate, well-designed, prospective clinical trials. Our choice of low-dose FK506 was based on data from preclinical studies (12) and the desire to avoid major immunosuppressive adverse effects in patients with indwelling lines. We did not observe an increase in line sepsis or opportunistic infections. We also did not observe serious adverse effects of posterior reversible encephalopathy syndrome, acute kidney injury or worsening of creatinine, an elevated systemic blood pressure, hyperglycemia, hyperkalemia, anemia, or a change in white blood cell count. The currently underway clinical phase IIa trial will address safety and tolerability in greater detail, as even low-dose immunosuppression over time can lead to complications. This is the first study in patients with PAH that repurposes FK506 to increase BMPR2 signaling. The changes in serologic biomarkers are encouraging and show that we have indeed targeted BMPR2 in patients with reduced levels of BMPR2. It will be of interest to determine whether the same effect can be achieved in patients with documented mutations and whether a subset of patients is particularly sensitive to the beneficial effects of this strategy and could therefore be identified up front as potential “responders” on the basis of BMPR2 levels.

Current Clinical Management of Pulmonary Arterial Hypertension

During the past 2 decades, there has been a tremendous evolution in the evaluation and care of patients with pulmonary arterial hypertension (PAH). The introduction of targeted PAH therapy consisting of prostacyclin and its analogs, endothelin antagonists, phosphodiesterase-5 inhibitors, and now a soluble guanylate cyclase activator have increased therapeutic options and potentially reduced morbidity and mortality; yet, none of the current therapies have been curative. Current clinical management of PAH has become more complex given the focus on early diagnosis, an increased number of available therapeutics within each mechanistic class, and the emergence of clinically challenging scenarios such as perioperative care. Efforts to standardize the clinical care of patients with PAH have led to the formation of multidisciplinary PAH tertiary care programs that strive to offer medical care based on peer-reviewed evidence-based, and expert consensus guidelines. Furthermore, these tertiary PAH centers often support clinical and basic science research programs to gain novel insights into the pathogenesis of PAH with the goal to improve the clinical management of this devastating disease. In this article, we discuss the clinical approach and management of PAH from the perspective of a single US-based academic institution. We provide an overview of currently available clinical guidelines and offer some insight into how we approach current controversies in clinical management of certain patient subsets. We conclude with an overview of our program structure and a perspective on research and the role of a tertiary PAH center in contributing new knowledge to the field.

Macrophages in solid organ transplantation

Macrophages are highly plastic hematopoietic cells with diversified functions related to their anatomic location and differentiation states. A number of recent studies have examined the role of macrophages in solid organ transplantation. These studies show that macrophages can induce allograft injury but, conversely, can also promote tissue repair in ischemia-reperfusion injury and acute rejection. Therapeutic strategies that target macrophages to improve outcomes in solid organ transplant recipients are being examined in preclinical and clinical models. In this review, we discuss the role of macrophages in different types of injury and rejection, with a focus on macrophage-mediated tissue injury, specifically vascular injury, repair and remodeling. We also discuss emerging macrophage-centered therapeutic opportunities in solid organ transplantation.

Leukotrienes in pulmonary arterial hypertension.

Abstract Leukotrienes (LTs) are lipid mediators derived from the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism and are markers and mediators of pulmonary inflammation. Research over the past two decades has established that LTs modulate inflammation in pulmonary arterial hypertension (PAH). The purpose of this review was to summarize the current knowledge of LTs in the pathophysiology of PAH and to highlight a recent study that advances our understanding of how leukotriene B4 (LTB4) specifically contributes to pulmonary vascular remodeling. The results of these studies suggest that pharmacological inhibition of LT pathways, especially LTB4, has high potential for the treatment of PAH.

Promotion of airway anastomotic microvascular regeneration and alleviation of airway ischemia by deferoxamine nanoparticles.

Airway tissue ischemia and hypoxia in human lung transplantation is a consequence of the sacrifice of the bronchial circulation during the surgical procedure and is a major risk factor for the development of airway anastomotic complications. Augmented expression of hypoxia-inducible factor (HIF)-1α promotes microvascular repair and alleviates allograft ischemia and hypoxia. Deferoxamine mesylate (DFO) is an FDA-approved iron chelator which has been shown to upregulate cellular HIF-1α. Here, we developed a nanoparticle formulation of DFO that can be topically applied to airway transplants at the time of surgery. In a mouse orthotopic tracheal transplant (OTT) model, the DFO nanoparticle was highly effective in enhancing airway microvascular perfusion following transplantation through the production of the angiogenic factors, placental growth factor (PLGF) and stromal cell-derived factor (SDF)-1. The endothelial cells in DFO treated airways displayed higher levels of p-eNOS and Ki67, less apoptosis, and decreased production of perivascular reactive oxygen species (ROS) compared to vehicle-treated airways. In summary, a DFO formulation topically-applied at the time of surgery successfully augmented airway anastomotic microvascular regeneration and the repair of alloimmune-injured microvasculature. This approach may be an effective topical transplant-conditioning therapy for preventing airway complications following clinical lung transplantation.

Leukotriene B4 Activates Pulmonary Artery Adventitial Fibroblasts in Pulmonary Hypertension.

A recent study demonstrated a significant role for leukotriene B4 (LTB4) causing pulmonary vascular remodeling in pulmonary arterial hypertension. LTB4 was found to directly injure luminal endothelial cells and promote growth of the smooth muscle cell layer of pulmonary arterioles. The purpose of this study was to determine the effects of LTB4 on the pulmonary adventitial layer, largely composed of fibroblasts. Here, we demonstrate that LTB4 enhanced human pulmonary artery adventitial fibroblast proliferation, migration, and differentiation in a dose-dependent manner through its cognate G-protein–coupled receptor, BLT1. LTB4 activated human pulmonary artery adventitial fibroblast by upregulating p38 mitogen-activated protein kinase as well as Nox4-signaling pathways. In an autoimmune model of pulmonary hypertension, inhibition of these pathways blocked perivascular inflammation, decreased Nox4 expression, reduced reactive oxygen species production, reversed arteriolar adventitial fibroblast activation, and attenuated pulmonary hypertension development. This study uncovers a novel mechanism by which LTB4 further promotes pulmonary arterial hypertension pathogenesis, beyond its established effects on endothelial and smooth muscle cells, by activating adventitial fibroblasts.

Graft microvascular disease in solid organ transplantation

Alloimmune inflammation damages the microvasculature of solid organ transplants during acute rejection. Although immunosuppressive drugs diminish the inflammatory response, they do not directly promote vascular repair. Repetitive microvascular injury with insufficient regeneration results in prolonged tissue hypoxia and fibrotic remodeling. While clinical studies show that a loss of the microvascular circulation precedes and may act as an initiating factor for the development of chronic rejection, preclinical studies demonstrate that improved microvascular perfusion during acute rejection delays and attenuates tissue fibrosis. Therefore, preservation of a functional microvasculature may represent an effective therapeutic strategy for preventing chronic rejection. Here, we review recent advances in our understanding of the role of the microvasculature in the long-term survival of transplanted solid organs. We also highlight microvessel-centered therapeutic strategies for prolonging the survival of solid organ transplants.